Compositions and methods for the therapy and diagnosis of influenza

ABSTRACT

The present invention provides novel human anti-influenza antibodies and related compositions and methods. These antibodies are used in the diagnosis and treatment of influenza infection.

RELATED APPLICATIONS

This application claims the benefit of provisional applications U.S.Ser. No. 60/987,353, filed Nov. 12, 2007, U.S. Ser. No. 60/987,355,filed Nov. 12, 2007, U.S. Ser. No. 61/053,840 filed May 16, 2008, andU.S. Ser. No. 61/095,208, filed Sep. 8, 2008, the contents which areeach herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to therapy, diagnosis andmonitoring of influenza infection. The invention is more specificallyrelated to methods of identifying influenza matrix 2 protein-specificantibodies and their manufacture and use. Such antibodies are useful inpharmaceutical compositions for the prevention and treatment ofinfluenza, and for the diagnosis and monitoring of influenza infection.

BACKGROUND OF THE INVENTION

Influenza virus infects 5-20% of the population and results in30,000-50,000 deaths each year in the U.S. Although the influenzavaccine is the primary method of infection prevention, four antiviraldrugs are also available in the U.S.: amantadine, rimantadine,oseltamivir and zanamivir. As of December 2005, only oseltamivir(TAMIFLU™) is recommended for treatment of influenza A due to theincreasing resistance of the virus to amantadine and rimantadineresulting from an amino acid substitution in the M2 protein of thevirus.

Disease caused by influenza A viral infections is typified by itscyclical nature. Antigenic drift and shift allow for different A strainsto emerge every year. Added to that, the threat of highly pathogenicstrains entering into the general population has stressed the need fornovel therapies for flu infections. The predominant fraction ofneutralizing antibodies is directed to the polymorphic regions of thehemagglutinin and neuraminidase proteins. Thus, such a neutralizing MAbwould presumably target only one or a few strains. A recent focus hasbeen on the relatively invariant matrix 2 (M2) protein. Potentially, aneutralizing MAb to M2 would be an adequate therapy for all influenza Astrains.

The M2 protein is found in a homotetramer that forms an ion channel andis thought to aid in the uncoating of the virus upon entering the cell.After infection, M2 can be found in abundance at the cell surface. It issubsequently incorporated into the virion coat, where it only comprisesabout 2% of total coat protein. The M2 extracellular domain (M2e) isshort, with the aminoterminal 2-24 amino acids displayed outside of thecell. Anti-M2 MAbs to date have been directed towards this linearsequence. Thus, they may not exhibit desired binding properties tocellularly expressed M2, including conformational determinants on nativeM2.

Therefore, a long-felt need exists in the art for new antibodies thatbind to the cell-expressed M2 and conformational determinants on thenative M2.

SUMMARY OF THE INVENTION

The present invention provides fully human monoclonal antibodiesspecifically directed against M2e. Optionally, the antibody is isolatedform a B-cell from a human donor. Exemplary monoclonal antibodiesinclude 8i10, 21B15 and 23K12 described herein. Alternatively, themonoclonal antibody is an antibody that binds to the same epitope as8i10, 21B15 and 23K12. The antibodies are respectively referred toherein is huM2e antibodies. The huM2e antibody has one or more of thefollowing characteristics: a) binds to an epitope in the extracellulardomain of the matrix 2 ectodomain (M2e) polypeptide of an influenzavirus; b) binds to influenza A infected cells; or c) binds to influenzaA virus.

The epitope that huM2e antibody binds to is a non-linear epitope of a M2polypeptide. Preferably, the epitope includes the amino terminal regionof the M2e polypeptide. More preferably the epitope wholly or partiallyincludes the amino acid sequence SLLTEV (SEQ ID NO:42). Most preferably,the epitope includes the amino acid at position 2, 5 and 6 of the M2epolypeptide when numbered in accordance with SEQ ID NO: 1. The aminoacid at position 2 is a serine; at position 5 is a threonine; and atposition 6 is a glutamic acid.

A huM2e antibody contains a heavy chain variable having the amino acidsequence of SEQ ID NOS: 44 or 50 and a light chain variable having theamino acid sequence of SEQ ID NOS: 46 or 52. Preferably, the three heavychain CDRs include an amino acid sequence at least 90%, 92%, 95%, 97%98%, 99% or more identical to the amino acid sequence of NYYWS (SEQ IDNO: 72), FIYYGGNTKYNPSLKS (SEQ ID NO: 74), ASCSGGYCILD (SEQ ID NO: 76),SNYMS (SEQ ID NO: 103), VIYSGGSTYYADSVK (SEQ ID NO: 105), CLSRMRGYGLDV(SEQ ID NO: 107) (as determined by the Kabat method) or ASCSGGYCILD (SEQID NO: 76), CLSRMRGYGLDV (SEQ ID NO: 107), GSSISN (SEQ ID NO: 109),FIYYGGNTK (SEQ ID NO: 110), GSSISN (SEQ ID NO: 111), GFTVSSN (SEQ ID NO:112), VIYSGGSTY (SEQ ID NO: 113) (as determined by the Chothia method)and a light chain with three CDRs that include an amino acid sequence atleast 90%, 92%, 95%, 97% 98%, 99% or more identical to the amino acidsequence of RASQNIYKYLN (SEQ ID NO: 59), AASGLQS (SEQ ID NO: 61),QQSYSPPLT (SEQ ID NO: 63), RTSQSISSYLN (SEQ ID NO: 92), AASSLQSGVPSRF(SEQ ID NO: 94), QQSYSMPA (SEQ ID NO: 96) (as determined by the Kabatmethod) or RASQNIYKYLN (SEQ ID NO: 59), AASGLQS (SEQ ID NO: 61),QQSYSPPLT (SEQ ID NO: 63), RTSQSISSYLN (SEQ ID NO: 92), AASSLQSGVPSRF(SEQ ID NO: 94), QQSYSMPA (SEQ ID NO: 96) (as determined by the Chothiamethod). The antibody binds M2e.

The heavy chain of a M2e antibody is derived from a germ line V(variable) gene such as, for example, the IgHV4 or the IgHV3 germlinegene.

The M2e antibodies of the invention include a variable heavy chain(V_(H)) region encoded by a human IgHV4 or the IgHV3 germline genesequence. A IgHV4 germline gene sequence are shown, e.g., in Accessionnumbers L10088, M29812, M95114, X56360 and M95117. IgHV3 germline genesequence are shown, e.g., in Accession numbers X92218, X70208, Z27504,M99679 and AB019437. The M2e antibodies of the invention include a V_(H)region that is encoded by a nucleic acid sequence that is at least 80%homologous to the IgHV4 or the IgHV3 germline gene sequence. Preferably,the nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous tothe IgHV4 or the IgHV3 germline gene sequence, and more preferably, atleast 98%, 99% homologous to the IgHV4 or the IgHV3 germline genesequence. The V_(H) region of the M2e antibody is at least 80%homologous to the amino acid sequence of the V_(H) region encoded by theIgHV4 or the IgHV3 V_(H) germline gene sequence. Preferably, the aminoacid sequence of V_(H) region of the M2e antibody is at least 90%, 95%,96%, 97% homologous to the amino acid sequence encoded by the IgHV4 orthe IgHV3 germline gene sequence, and more preferably, at least 98%, 99%homologous to the sequence encoded by the IgHV4 or the IgHV3 germlinegene sequence.

The M2e antibodies of the invention also include a variable light chain(V_(L)) region encoded by a human IgKV1 germline gene sequence. A humanIgKV1 V_(L) germline gene sequence is shown, e.g., Accession numbersX59315, X59312, X59318, J00248, and Y14865. Alternatively, the M2eantibodies include a V_(L) region that is encoded by a nucleic acidsequence that is at least 80% homologous to the IgKV1 germline genesequence. Preferably, the nucleic acid sequence is at least 90%, 95%,96%, 97% homologous to the IgKV1 germline gene sequence, and morepreferably, at least 98%, 99% homologous to the IgKV1 germline genesequence. The V_(L) region of the M2e antibody is at least 80%homologous to the amino acid sequence of the V_(L) region encoded theIgKV1 germline gene sequence. Preferably, the amino acid sequence ofV_(L) region of the M2e antibody is at least 90%, 95%, 96%, 97%homologous to the amino acid sequence encoded by the IgKV1 germline genesequence, and more preferably, at least 98%, 99% homologous to thesequence encoded by e the IgKV1 germline gene sequence.

In another aspect the invention provides a composition including anhuM2e antibody according to the invention. In various aspects thecomposition further includes an anti-viral drug, a viral entry inhibitoror a viral attachment inhibitor. The anti-viral drug is for example aneuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor or anM2 ion channel inhibitor. The M2 ion channel inhibitor is for exampleamantadine or rimantadine. The neuraminidase inhibitor for examplezanamivir, or oseltamivir phosphate. In a further aspect the compositionfurther includes a second anti-influenza A antibody.

In a further aspect the huM2e antibodies according to the invention areoperably-linked to a therapeutic agent or a detectable label.

Additionally, the invention provides methods for stimulating an immuneresponse, treating, preventing or alleviating a symptom of an influenzaviral infection by administering an huM2e antibody to a subject

Optionally, the subject is further administered with a second agent suchas, but not limited to, an influenza virus antibody, an anti-viral drugsuch as a neuraminidase inhibitor, a HA inhibitor, a sialic acidinhibitor or an M2 ion channel inhibitor, a viral entry inhibitor or aviral attachment inhibitor. The M2 ion channel inhibitor is for exampleamantadine or rimantadine. The neuraminidase inhibitor for examplezanamivir, or oseltamivir phosphate. The subject is suffering from or ispredisposed to developing an influenza virus infection, such as, forexample, an autoimmune disease or an inflammatory disorder.

In another aspect, the invention provides methods of administering thehuM2e antibody of the invention to a subject prior to, and/or afterexposure to an influenza virus. For example, the huM2e antibody of theinvention is used to treat or prevent rejection influenza infection. ThehuM2e antibody is administered at a dose sufficient to promote viralclearance or eliminate influenza A infected cells.

Also included in the invention is a method for determining the presenceof an influenza virus infection in a patient, by contacting a biologicalsample obtained from the patient with a humM2e antibody; detecting anamount of the antibody that binds to the biological sample; andcomparing the amount of antibody that binds to the biological sample toa control value.

The invention further provides a diagnostic kit comprising a huM2eantibody.

Other features and advantages of the invention will be apparent from andare encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the binding of three antibodies of the present inventionand control hu14C2 antibody to 293-HEK cells transfected with an M2expression construct or control vector, in the presence or absence offree M2 peptide.

FIGS. 2A and B are graphs showing human monoclonal antibody binding toinfluenza A/Puerto Rico/8/32.

FIG. 3A is a chart showing amino acid sequences of extracellular domainsof M2 variants.

FIGS. 3B and C are bar charts showing binding of human monoclonalanti-influenza antibody binding to M2 variants shown in FIG. 3A.

FIGS. 4A and B are bar charts showing binding of human monoclonalanti-influenza antibody binding to M2 peptides subjected to alaninescanning mutagenesis.

FIG. 5 is a series of bar charts showing binding of MAbs 8i10 and 23K12to M2 protein representing influenza strain A/HK/483/1997 sequence thatwas stably expressed in the CHO cell line DG44.

FIG. 6A is a chart showing cross reactivity binding of anti-M2antibodies to variant M2 peptides.

FIG. 6B is a chart showing binding activity of M2 antibodies totruncated M2 peptides.

FIG. 7 is a graph showing survival of influenza infected mice treatedwith human anti-influenza monoclonal antibodies.

FIG. 8 is an illustration showing the anti-M2 antibodies bind a highlyconserved region in the N-Terminus of M2e.

FIG. 9 is a graph showing anti-M2 rHMAb clones from crude supernatantbound to influenza on ELISA, whereas the control anti-M2e mAb 14C2 didnot readily bind virus.

FIG. 10 is a series of photographs showing anti-M2 rHMAbs bound to cellsinfected with influenza. MDCK cells were or were not infected withinfluenza A/PR/8/32 and Ab binding from crude supernatant was tested 24hours later. Data were gathered from the FMAT plate scanner.

FIG. 11 is a graph showing anti-M2 rHMAb clones from crude supernatantbound to cells transfected with the influenza subtypes H₃N₂, HK483, andVN1203 M2 proteins. Plasmids encoding full length M2 cDNAs correspondingto influenza strains H₃N₂, HK483, and VN1203, as well as a mock plasmidcontrol, were transiently transfected into 293 cells. The 14C2, 8i10,23K12, and 21B15 mABs were tested for binding to the transfectants, andwere detected with an AF647-conjugated anti-human IgG secondaryantibody. Shown are the mean fluorescence intensities of the specificmAB bound after FACS analysis.

DETAILED DESCRIPTION

The present invention provides fully human monoclonal antibodiesspecific against the extracellular domain of the matrix 2 (M2)polypeptide. The antibodies are respectively referred to herein is huM2eantibodies.

M2 is a 96 amino acid transmembrane protein present as a homotetramer onthe surface of influenza virus and virally infected cells. M2 contains a23 amino acid ectodomain (M2e) that is highly conserved across influenzaA strains. Few amino acid changes have occurred since the 1918 pandemicstrain thus M2e is an attractive target for influenza therapies. Inprior studies, monoclonal antibodies specific to the M2 ectodomain (M2e)were derived upon immunizations with a peptide corresponding to thelinear sequence of M2e. In contrast, the present invention provides anovel process whereby full-length M2 is expressed in cell lines, whichallows for the identification of human antibodies that bound thiscell-expressed M2e. The huM2e antibodies have been shown to bindconformational determinants on the M2-transfected cells, as well asnative M2, either on influenza infected cells, or on the virus itself.The huM2e antibodies did not bind the linear M2e peptide, but they dobind several natural M2 variants, also expressed upon cDNA transfectioninto cell lines. Thus, this invention has allowed for the identificationand production of human monoclonal antibodies that exhibit novelspecificity for a very broad range of influenza A virus strains. Theseantibodies may be used diagnostically to identify influenza A infectionand therapeutically to treat influenza A infection.

The huM2e antibodies of the invention have one or more of the followingcharacteristics: the huM2e antibody binds a) to an epitope in theextracellular domain of the matrix 2 (M2) polypeptide of an influenzavirus; b) binds to influenza A infected cells; and/or c) binds toinfluenza A virus (i.e., virons). The huM2e antibodies of the inventioneliminate influenza infected cells through immune effector mechanismssuch as ADCC and promotes direct viral clearance by binding to influenzavirons. The huM2e antibodies of the invention bind to the amino-terminalregion of the M2e polypeptide. Preferably, the huM2e antibodies of theinvention bind to the amino-terminal region of the M2e polypeptidewherein the N-terminal methionine residue is absent. Exemplary M2esequences include those sequences listed on Table I below

TABLE I Type Name Subtype M2E Sequence SEQ ID NO A BREVIG H1N1MSLLTEVETPTRNEWGCRCNDSSD SEQ ID NO: 1 MISSION.1.1918 A FORTMONMOUTH.1.1947 H1N1 MSLLTEVETPTKNEWECRCNDSSD SEQ ID NO: 2 A.SINGAPORE.02.2005 H3N2 MSLLTEVETPIRNEWECRCNDSSD SEQ ID NO: 3 AWISCONSIN.10.98 H1N1 MSLLTEVETPIRNGWECKCNDSSD SEQ ID NO: 4 AWISCONSIN.301.1976 H1N1 MSLLTEVETPIRSEWGCRCNDSSD SEQ ID NO: 5 APANAMA.1.66 H2N2 MSFLPEVETPIRNEWGCRCNDSSD SEQ ID NO: 6 A NEWYORK.321.1999 H3N2 MSLLTEVETPIRNEWGCRCNDSSN SEQ ID NO: 7 A CARACAS.1.71H3N2 MSLLTEVETPIRKEWGCRCNDSSD SEQ ID NO: 8 A TAIWAN.3.71 H3N2MSFLTEVETPIRNEWGCRCNDSSD SEQ ID NO: 9 A WUHAN.359.95 H3N2MSLPTEVETPIRSEWGCRCNDSSD SEQ ID NO: 10 A HONG KONG.1144.99 H3N2MSLLPEVETPIRNEWGCRCNDSSD SEQ ID NO: 11 A HONG KONG.1180.99 H3N2MSLLPEVETPIRNGWGCRCNDSSD SEQ ID NO: 12 A HONG KONG.1774.99 H3N2MSLLTEVETPTRNGWECRCSGSSD SEQ ID NO: 13 A NEW YORK.217.02 H1N2MSLLTEVETPIRNEWEYRCNDSSD SEQ ID NO: 14 A NEW YORK.300.2003 H1N2MSLLTEVETPIRNEWEYRCSDSSD SEQ ID NO: 15 A SWINE.SPAIN.54008.2004 H3N2MSLLTEVETPTRNGWECRYSDSSD SEQ ID NO: 16 A GUANGZHOU.333.99 H9N2MSFLTEVETLTRNGWECRCSDSSD SEQ ID NO: 17 A HONG KONG.1073.99 H9N2MSLLTEVETLTRNGWECKCRDSSD SEQ ID NO: 18 A HONG KONG.1.68 H3N2MSLLTEVETPIRNEWGCRCNDSSD SEQ ID NO: 19 A SWINE.HONG H3N2MSLLTEVETPIRSEWGCRCNDSGD SEQ ID NO: 20 KONG.126.1982 A NEW YORK.703.1995H3N2 MSLLTEVETPIRNEWECRCNGSSD SEQ ID NO: 21 A SWINE.QUEBEC.192.81 H1N1MSLPTEVETPIRNEWGCRCNDSSD SEQ ID NO: 22 A PUERTO RICO.8.34 H1N1MSLLTEVETPIRNEWGCRCNGSSD SEQ ID NO: 23 A HONG KONG.485.97 HEN1MSLLTEVDTLTRNGWGCRCSDSSD SEQ ID NO: 24 A HONG KONG.542.97 HEN1MSLLTEVETLTKNGWGCRCSDSSD SEQ ID NO: 25 A SILKY H9N2MSLLTEVETPTRNGWECKCSDSSD SEQ ID NO: 26 CHICKEN.SHANTOU.1826 .2004 ACHICKEN.TAIWAN.0305.04 H6N1 MSLLTEVETHTRNGWECKCSDSSD SEQ ID NO: 27 AQUAIL.ARKANSAS.16309-7.94 H7N3NSA MSLLTEVKTPTRNGWECKCSDSSD SEQ ID NO: 28A HONG KONG.486.97 HEN1 MSLLTEVETLTRNGWGCRCSDSSD SEQ ID NO: 29 ACHICKEN.PENNSYLVANIA H7N2NSB MSLLTEVETPTRDGWECKCSDSSD SEQ ID NO: 30.13552-1.98 A CHICKEN.HEILONGJIANG H9N2 MSLLTEVETPTRNGWGCRCSDSSD SEQ IDNO: 31 .48.01 A SWINE.KOREA.55.2005 H1N2 MSLLTEVETPTRNGWECKCNDSSD SEQ IDNO: 32 A HONG KONG.1073.99 H9N2 MSLLTEVETLTRNGWECKCSDSSD SEQ ID NO: 33 AWISCONSIN.3523.88 H1N1 MSLLTEVETPIRNEWGCKCNDSSD SEQ ID NO: 34 A X-31VACCINE STRAIN H3N2 MSFLTEVETPIRNEWGCRCNGSSD SEQ ID NO: 35 ACHICKEN.ROSTOCK.8.19 H7N1 MSLLTEVETPTRNGWECRCNDSSD SEQ ID NO: 36 34 AENVIRONMENT.NEW H7N2 MSLLTEVETPIRKGWECNCSDSSD SEQ ID NO: 37YORK.16326-1.2005 A INDONESIA.560H.2006 HEN1 MSLLTEVETPTRNEWECRCSDSSDSEQ ID NO: 38 A CHICKEN.HONG H9N2 MSLLTGVETHTRNGWGCKCSDSSD SEQ ID NO: 39KONG.SF1.03 A CHICKEN.HONGKONG.YU4 H9N2 MSLLPEVETHTRNGWGCRCSDSSD SEQ IDNO: 40 27.03

In one embodiment, the huM2e antibodies of the invention bind to a M2ethat wholly or partially includes the amino acid residues from position2 to position 7 of M2e when numbered in accordance with SEQ ID NO:1. Forexample, the huM2e antibodies of the invention bind wholly or partiallyto the amino acid sequence SLLTEVET (SEQ ID NO: 41) Most preferably, thehuM2e antibodies of the invention bind wholly or partially to the aminoacid sequence SLLTEV (SEQ ID NO: 42) Preferably, the huM2e antibodies ofthe invention bind to non-linear epitope of the M2e protein. Forexample, the huM2e antibodies bind to an epitope comprising position 2,5, and 6 of the M2e polypeptide when numbered in accordance to SEQ IDNO: 1 where the amino acid at a) position 2 is a serine; b) position 5is a threonine; and c) position 6 is a glutamic acid. Exemplary huM2emonoclonal antibodies that binds to this epitope are the 8I10, 21B15 or23K12 antibodies described herein.

The 8I10 antibody includes a heavy chain variable region (SEQ ID NO: 44)encoded by the nucleic acid sequence shown below in SEQ ID NO: 43, and alight chain variable region (SEQ ID NO: 46) encoded by the nucleic acidsequence shown in SEQ ID NO: 45.

The amino acids encompassing the CDRs as defined by Chothia, C. et al.(1989, Nature, 342: 877-883) are underlined and those defined by KabatE. A. et al. (1991, Sequences of Proteins of Immunological Interest,5^(th) edit., NIH Publication no. 91-3242 U.S. Department of Heath andHuman Services.) are highlighted in bold in the sequences below.

The heavy chain CDRs of the 8I10 antibody have the following sequencesper Kabat definition: NYYWS (SEQ ID NO: 72), FIYYGGNTKYNPS LKS (SEQ IDNO: 74) and ASCSGGYCILD (SEQ ID NO: 76). The light chain CDRs of the8I10 antibody have the following sequences per Kabat definition:RASQNIYKYLN (SEQ ID NO: 59), AA SGLQS (SEQ ID NO: 61) and QQSYSPPLT (SEQID NO: 63).

The heavy chain CDRs of the 8I10 antibody have the following sequencesper Chothia definition: GSSISN (SEQ ID NO: 109), FIYYGGNTK (SEQ ID NO:110) and ASCSGGYCILD (SEQ ID NO: 76). The light chain CDRs of the 8I10antibody have the following sequences per Chothia definition:RASQNIYKYLN (SEQ ID NO: 59), AASGLQS (SEQ ID NO: 61) and QQSYSPPLT (SEQID NO: 63).

>8110 VII nucleotide sequence: (SEQ ID NO: 43)CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGTAGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCG >8110 VIIamino acid sequence: (SEQ ID NO: 44) Kabat Bold, Chothia underlined Q VQ L Q E S G P G L V K P S E T L S L T C T V S G S S I S N  Y Y W S W I RQ S P G K G L E W I G F I Y Y G G N T K  Y N P S L K S R V T I S Q D T SK S Q V S L T M S S V T A A E S A V Y F C A R A S C S G G Y C I L D  Y WG Q G T L V T V S >8110 VL nucleotide sequence: (SEQ ID NO: 45)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCCCCTAAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGGTGGAGATCAAAC >8110 VL amino acid sequence: (SEQ ID NO: 46)Kabat Bold, Chothia underlined D I Q M T Q S P S S L S A S V G D R V T IT C R A S Q N I Y K Y L N  W Y Q Q R P G K A P K G L I S A A S G L Q S G V P S R E S G S G S G T D F T L T I T S L Q P E D F A T Y Y CQ Q S Y S P P L T  F G G G T R V E I K

The 21B15 antibody includes antibody includes a heavy chain variableregion (SEQ ID NO: 44) encoded by the nucleic acid sequence shown belowin SEQ ID NO: 47, and a light chain variable region (SEQ ID NO: 46)encoded by the nucleic acid sequence shown in SEQ ID NO: 48.

The amino acids encompassing the CDRs as defined by Chothia et al. 1989,are underlined and those defined by Kabat et al., 1991 are highlightedin bold in the sequences below.

The heavy chain CDRs of the 21B15 antibody have the following sequencesper Kabat definition: NYYWS (SEQ ID NO: 72), FIYYGGNTKYNPSLKS (SEQ IDNO: 74) and ASCSGGYCILD (SEQ ID NO: 76). The light chain CDRs of the21B15 antibody have the following sequences per Kabat definition:RASQNIYKYLN (SEQ ID NO: 59), AASGLQS (SEQ ID NO: 61) and QQSYSPPLT (SEQID NO: 63).

The heavy chain CDRs of the 21B15 antibody have the following sequencesper Chothia definition: GSSISN (SEQ ID NO: 111), FIYYGGNTK (SEQ ID NO:110) and ASCSGGYCILD (SEQ ID NO: 76). The light chain CDRs of the 21B15antibody have the following sequences per Chothia definition:RASQNIYKYLN (SEQ ID NO: 59), AASGLQS (SEQ ID NO: 61) and QQSYSPPLT (SEQID NO: 63).

>21B15 VII nucleotide sequence: (SEQ ID NO: 47)CAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAGTACAATCCCTCCCTCAAGAGCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGCTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGTAGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCG >21B15 VIIamino acid sequence: (SEQ ID NO: 44) Kabat Bold, Chothia underlined Q VQ L Q E S G P G L V K P S E T L S L T C T V S G S S I S N  Y Y W S W I RQ S P G K G L E W I G F I Y Y G G N T K  Y N P S L K S R V T I S Q D T SK S Q V S L T M S S V T A A E S A V Y F C A R A S C S G G Y C I L D  Y WG Q G T L V T V S >21B15 VL nucleotide sequence: (SEQ ID NO: 48)GACATCCAGGTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGCGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCCCCTAAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGGTGGATATCAAAC >2221B15 VL amino acid sequence: (SEQ ID NO: 46)Kabat Bold, Chothia underlined D I Q V T Q S P S S L S A S V G D R V T IT C R A S Q N I Y K Y L N  W Y Q Q R P G K A P K G L I S A A S G L Q S G V P S R F S G S G S G T D F T L T I T S L Q P E D F A T Y Y CQ Q S Y S P P L  T F G G G T R V D I K

The 23K12 antibody includes antibody includes a heavy chain variableregion (SEQ ID NO: 50) encoded by the nucleic acid sequence shown belowin SEQ ID NO: 49, and a light chain variable region (SEQ ID NO: 52)encoded by the nucleic acid sequence shown in SEQ ID NO: 51.

The amino acids encompassing the CDRs as defined by Chothia et al., 1989are underlined and those defined by Kabat et al., 1991 are highlightedin bold in the sequences below.

The heavy chain CDRs of the 23K12 antibody have the following sequencesper Kabat definition: SNYMS (SEQ ID NO: 103), VIYSGGSTYYADSVK (SEQ IDNO: 105) and CLSRMRGYGLDV (SEQ ID NO: 107). The light chain CDRs of the23K12 antibody have the following sequences per Kabat definition:RTSQSISSYLN (SEQ ID NO: 92), AASSLQSGVPSRF (SEQ ID NO: 94) and QQSYSMPA(SEQ ID NO: 96).

The heavy chain CDRs of the 23K12 antibody have the following sequencesper Chothia definition: GFTVSSN (SEQ ID NO: 112), VIYSGGSTY (SEQ ID NO:113) and CLSRMRGYGLDV (SEQ ID NO: 107). The light chain CDRs of the23K12 antibody have the following sequences per Chothia definition:RTSQSISSYLN (SEQ ID NO: 92), AASSLQSGVPSRF (SEQ ID NO: 94) and QQSYSMPA(SEQ ID NO: 96).

>23K12 VII nucleotide sequence: (SEQ ID NO: 49)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGAATCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTTATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCAGATTCTCCTTCTCCAGAGACAACTCCAAGAACACAGTGTTTCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATGTCTGAGCAGGATGCGGGGTTACGGTTTAGACGTCTGGGGCCAAGGGACCACGGTCAC CGTCTCG >2223K12 VIIamino acid sequence: (SEQ ID NO: 50) Kabat Bold, Chothia underlined E VQ L V E S G G G L V Q P G G S L R I S C A A S G F T V S S N  Y M S W V RQ A P G K G L E W V S V I Y S G G S T Y  Y A D S V K G R F S F S R D N SK N T V F L Q M N S L R A E D T A V Y Y C A R C L S R M R G Y G L D V  WG Q G T T V T V S >23K12 VL nucleotide sequence: (SEQ ID NO: 51)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGACAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGGTCTGCAACCTGAAGATTTTGCAACCTACTACTGTCAACAGAGTTACAGTATGCCTGCCTTTGGCCAGGGGACCAAGCTGGAGATCAAA >23K12 VL amino acid sequence: (SEQ ID NO: 52) KabatBold, Chothia underlined D I Q M T Q S P S S L S A S V G D R V T I T CR T S Q S I S S Y L N  W Y Q Q K P G K A P K L L I YA A S S L Q S G V P S R F  S G S G S G T D F T L T I S G L Q P E D F A TY Y C Q Q S Y S M P A  F G Q G T K L E I K

HuM2e antibodies of the invention also include antibodies that include aheavy chain variable amino acid sequence that is at least 90%, 92%, 95%,97% 98%, 99% or more identical the amino acid sequence of SEQ ID NO: 44or 49. and/or a light chain variable amino acid that is at least 90%,92%, 95%, 97% 98%, 99% or more identical the amino acid sequence of SEQID NO: 46 or 52.

Alternatively, the monoclonal antibody is an antibody that binds to thesame epitope as 8I10, 21B15 or 23K12.

The heavy chain of a M2e antibody is derived from a germ line V(variable) gene such as, for example, the IgHV4 or the IgHV3 germlinegene.

The M2e antibodies of the invention include a variable heavy chain(V_(H)) region encoded by a human IgHV4 or the IgHV3 germline genesequence. A IgHV4 germline gene sequence are shown, e.g., in Accessionnumbers L10088, M29812, M95114, X56360 and M95117. IgHV3 germline genesequence are shown, e.g., in Accession numbers X92218, X70208, Z27504,M99679 and AB019437. The M2e antibodies of the invention include a V_(H)region that is encoded by a nucleic acid sequence that is at least 80%homologous to the IgHV4 or the IgHV3 germline gene sequence. Preferably,the nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous tothe IgHV4 or the IgHV3 germline gene sequence, and more preferably, atleast 98%, 99% homologous to the IgHV4 or the IgHV3 germline genesequence. The V_(H) region of the M2e antibody is at least 80%homologous to the amino acid sequence of the V_(H) region encoded by theIgHV4 or the IgHV3 V_(H) germline gene sequence. Preferably, the aminoacid sequence of V_(H) region of the M2e antibody is at least 90%, 95%,96%, 97% homologous to the amino acid sequence encoded by the IgHV4 orthe IgHV3 germline gene sequence, and more preferably, at least 98%, 99%homologous to the sequence encoded by the IgHV4 or the IgHV3 germlinegene sequence.

The M2e antibodies of the invention also include a variable light chain(V_(L)) region encoded by a human IgKV1 germline gene sequence. A humanIgKV1 V_(L) germline gene sequence is shown, e.g., Accession numbersX59315, X59312, X59318, J00248, and Y14865. Alternatively, the M2eantibodies include a V_(L) region that is encoded by a nucleic acidsequence that is at least 80% homologous to the IgKV1 germline genesequence. Preferably, the nucleic acid sequence is at least 90%, 95%,96%, 97% homologous to the IgKV1 germline gene sequence, and morepreferably, at least 98%, 99% homologous to the IgKV1 germline genesequence. The V_(L) region of the M2e antibody is at least 80%homologous to the amino acid sequence of the V_(L) region encoded theIgKV1 germline gene sequence. Preferably, the amino acid sequence ofV_(L) region of the M2e antibody is at least 90%, 95%, 96%, 97%homologous to the amino acid sequence encoded by the IgKV1 germline genesequence, and more preferably, at least 98%, 99% homologous to thesequence encoded by e the IgKV1 germline gene sequence.

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The practice of the present invention will employ, unlessindicated specifically to the contrary, conventional methods ofvirology, immunology, microbiology, molecular biology and recombinantDNA techniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Sambrook, et al. Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984).

The nomenclatures utilized in connection with, and the laboratoryprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art. Standard techniquesare used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.

The following definitions are useful in understanding the presentinvention:

The term “antibody” (Ab) as used herein includes monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity. The term “immunoglobulin” (Ig) is usedinterchangeably with “antibody” herein.

An “isolated antibody” is one that has been separated and/or recoveredfrom a component of its natural environment. Contaminant components ofits natural environment are materials that would interfere withdiagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or nonproteinaceous solutes.In preferred embodiments, the antibody is purified: (1) to greater than95% by weight of antibody as determined by the Lowry method, and mostpreferably more than 99% by weight; (2) to a degree sufficient to obtainat least 15 residues of N-terminal or internal amino acid sequence byuse of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGEunder reducing or non-reducing conditions using Coomassie blue or,preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

The basic four-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. An IgM antibody consists of 5 of the basic heterotetramer unitalong with an additional polypeptide called J chain, and thereforecontain 10 antigen binding sites, while secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain. In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and ε isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain (C_(L)) at its other end. The V_(L) is aligned with the V_(H) andthe C_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see, e.g., Basic and Clinical Immunology, 8th edition,Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton& Lange, Norwalk, Conn., 1994, page 71, and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa (κ) and lambda (λ), based on theamino acid sequences of their constant domains (C_(L)). Depending on theamino acid sequence of the constant domain of their heavy chains(C_(H)), immunoglobulins can be assigned to different classes orisotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG,and IgM, having heavy chains designated alpha (

), delta (λ), epsilon (

), gamma (γ) and mu (μ), respectively. The γ and

classes are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term “variable” refers to the fact that certain segments of the Vdomains differ extensively in sequence among antibodies. The V domainmediates antigen binding and defines specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 110-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aβ-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody that are responsible for antigen binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g., around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the V_(H) when numberedin accordance with the Kabat numbering system; Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)); and/or thoseresidues from a “hypervariable loop” (e.g., residues 24-34 (L1), 50-56(L2) and 89-97 (L3) in the V_(L), and 26-32 (H1), 52-56 (H2) and 95-101(H3) in the V_(H) when numbered in accordance with the Chothia numberingsystem; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and/orthose residues from a “hypervariable loop”/CDR (e.g., residues 27-38(L1), 56-65 (L2) and 105-120 (L3) in the V_(L), and 27-38 (H1), 56-65(H2) and 105-120 (H3) in the V_(H) when numbered in accordance with theIMGT numbering system; Lefranc, M. P. et al. Nucl. Acids Res. 27:209-212(1999), Ruiz, M. e al. Nucl. Acids Res. 28:219-221 (2000)). Optionallythe antibody has symmetrical insertions at one or more of the followingpoints 28, 36 (L1), 63, 74-75 (L2) and 123 (L3) in the V_(L), and 28, 36(H1), 63, 74-75 (H2) and 123 (H3) in the V_(H) when numbered inaccordance with AHo; Honneger, A. and Plunkthun, A. J. Mol. Biol.309:657-670 (2001)).

By “germline nucleic acid residue” is meant the nucleic acid residuethat naturally occurs in a germline gene encoding a constant or variableregion. “Germline gene” is the DNA found in a germ cell (i.e., a celldestined to become an egg or in the sperm). A “germline mutation” refersto a heritable change in a particular DNA that has occurred in a germcell or the zygote at the single-cell stage, and when transmitted tooffspring, such a mutation is incorporated in every cell of the body. Agermline mutation is in contrast to a somatic mutation which is acquiredin a single body cell. In some cases, nucleotides in a germline DNAsequence encoding for a variable region are mutated (i.e., a somaticmutation) and replaced with a different nucleotide.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations that include different antibodies directed againstdifferent determinants (epitopes), each monoclonal antibody is directedagainst a single determinant on the antigen. In addition to theirspecificity, the monoclonal antibodies are advantageous in that they maybe synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies useful in the present invention may be prepared by thehybridoma methodology first described by Kohler et al., Nature, 256:495(1975), or may be made using recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The present inventionprovides variable domain antigen-binding sequences derived from humanantibodies. Accordingly, chimeric antibodies of primary interest hereininclude antibodies having one or more human antigen binding sequences(e.g., CDRs) and containing one or more sequences derived from anon-human antibody, e.g., an FR or C region sequence. In addition,chimeric antibodies of primary interest herein include those comprisinga human variable domain antigen binding sequence of one antibody classor subclass and another sequence, e.g., FR or C region sequence, derivedfrom another antibody class or subclass. Chimeric antibodies of interestherein also include those containing variable domain antigen-bindingsequences related to those described herein or derived from a differentspecies, such as a non-human primate (e.g., Old World Monkey, Ape, etc).Chimeric antibodies also include primatized and humanized antibodies.

Furthermore, chimeric antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

A “humanized antibody” is generally considered to be a human antibodythat has one or more amino acid residues introduced into it from asource that is non-human. These non-human amino acid residues are oftenreferred to as “import” residues, which are typically taken from an“import” variable domain. Humanization is traditionally performedfollowing the method of Winter and co-workers (Jones et al., Nature,321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988);Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting importhypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species.

A “human antibody” is an antibody containing only sequences present inan antibody naturally produced by a human. However, as used herein,human antibodies may comprise residues or modifications not found in anaturally occurring human antibody, including those modifications andvariant sequences described herein. These are typically made to furtherrefine or enhance antibody performance.

An “intact” antibody is one that comprises an antigen-binding site aswell as a C_(L) and at least heavy chain constant domains, C_(H)1,C_(H)2 and C_(H)3. The constant domains may be native sequence constantdomains (e.g., human native sequence constant domains) or amino acidsequence variant thereof. Preferably, the intact antibody has one ormore effector functions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870;Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments.

The phrase “functional fragment or analog” of an antibody is a compoundhaving qualitative biological activity in common with a full-lengthantibody. For example, a functional fragment or analog of an anti-IgEantibody is one that can bind to an IgE immunoglobulin in such a mannerso as to prevent or substantially reduce the ability of such moleculefrom having the ability to bind to the high affinity receptor, Fc_(ε)RI.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (C_(H) 1). Each Fab fragment is monovalent with respect toantigen binding, i.e., it has a single antigen-binding site. Pepsintreatment of an antibody yields a single large F(ab′)₂ fragment thatroughly corresponds to two disulfide linked Fab fragments havingdivalent antigen-binding activity and is still capable of cross-linkingantigen. Fab′ fragments differ from Fab fragments by having additionalfew residues at the carboxy terminus of the C_(H)1 domain including oneor more cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments that have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “Fc” fragment comprises the carboxy-terminal portions of both Hchains held together by disulfides. The effector functions of antibodiesare determined by sequences in the Fc region, which region is also thepart recognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (three loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

As used herein, an antibody that “internalizes” is one that is taken upby (i.e., enters) the cell upon binding to an antigen on a mammaliancell (e.g., a cell surface polypeptide or receptor). The internalizingantibody will of course include antibody fragments, human or chimericantibody, and antibody conjugates. For certain therapeutic applications,internalization in vivo is contemplated. The number of antibodymolecules internalized will be sufficient or adequate to kill a cell orinhibit its growth, especially an infected cell. Depending on thepotency of the antibody or antibody conjugate, in some instances, theuptake of a single antibody molecule into the cell is sufficient to killthe target cell to which the antibody binds. For example, certain toxinsare highly potent in killing such that internalization of one moleculeof the toxin conjugated to the antibody is sufficient to kill theinfected cell.

As used herein, an antibody is said to be “immunospecific,” “specificfor” or to “specifically bind” an antigen if it reacts at a detectablelevel with the antigen, preferably with an affinity constant, K_(a), ofgreater than or equal to about 10⁴ M⁻¹, or greater than or equal toabout 10⁵ M⁻¹, greater than or equal to about 10⁶ M⁻¹, greater than orequal to about 10⁷ M⁻¹, or greater than or equal to 10⁸ M⁻¹. Affinity ofan antibody for its cognate antigen is also commonly expressed as adissociation constant K_(D), and in certain embodiments, HuM2e antibodyspecifically binds to M2e if it binds with a K_(D) of less than or equalto 10⁻⁴ M, less than or equal to about 10⁻⁵ M, less than or equal toabout 10⁻⁶ M, less than or equal to 10⁻⁷ M, or less than or equal to10⁻⁸ M. Affinities of antibodies can be readily determined usingconventional techniques, for example, those described by Scatchard etal. (Ann. N.Y. Acad. Sci. USA 51:660 (1949)).

Binding properties of an antibody to antigens, cells or tissues thereofmay generally be determined and assessed using immunodetection methodsincluding, for example, immunofluorescence-based assays, such asimmuno-histochemistry (IHC) and/or fluorescence-activated cell sorting(FACS).

An antibody having a “biological characteristic” of a designatedantibody is one that possesses one or more of the biologicalcharacteristics of that antibody which distinguish it from otherantibodies. For example, in certain embodiments, an antibody with abiological characteristic of a designated antibody will bind the sameepitope as that bound by the designated antibody and/or have a commoneffector function as the designated antibody.

The term “antagonist” antibody is used in the broadest sense, andincludes an antibody that partially or fully blocks, inhibits, orneutralizes a biological activity of an epitope, polypeptide, or cellthat it specifically binds. Methods for identifying antagonistantibodies may comprise contacting a polypeptide or cell specificallybound by a candidate antagonist antibody with the candidate antagonistantibody and measuring a detectable change in one or more biologicalactivities normally associated with the polypeptide or cell.

An “antibody that inhibits the growth of infected cells” or a “growthinhibitory” antibody is one that binds to and results in measurablegrowth inhibition of infected cells expressing or capable of expressingan M2e epitope bound by an antibody. Preferred growth inhibitoryantibodies inhibit growth of infected cells by greater than 20%,preferably from about 20% to about 50%, and even more preferably, bygreater than 50% (e.g., from about 50% to about 100%) as compared to theappropriate control, the control typically being infected cells nottreated with the antibody being tested. Growth inhibition can bemeasured at an antibody concentration of about 0.1 to 30 μg/ml or about0.5 nM to 200 nM in cell culture, where the growth inhibition isdetermined 1-10 days after exposure of the infected cells to theantibody. Growth inhibition of infected cells in vivo can be determinedin various ways known in the art. The antibody is growth inhibitory invivo if administration of the antibody at about 1 μg/kg to about 100mg/kg body weight results in reduction the percent of infected cells ortotal number of infected cells within about 5 days to 3 months from thefirst administration of the antibody, preferably within about 5 to 30days.

An antibody that “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies).Preferably the cell is an infected cell. Various methods are availablefor evaluating the cellular events associated with apoptosis. Forexample, phosphatidyl serine (PS) translocation can be measured byannexin binding; DNA fragmentation can be evaluated through DNAladdering; and nuclear/chromatin condensation along with DNAfragmentation can be evaluated by any increase in hypodiploid cells.Preferably, the antibody that induces apoptosis is one that results inabout 2 to 50 fold, preferably about 5 to 50 fold, and most preferablyabout 10 to 50 fold, induction of annexin binding relative to untreatedcell in an annexin binding assay.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: Clq bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are required for such killing. The primary cells for mediatingADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI,FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarizedin Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92(1991). To assess ADCC activity of a molecule of interest, an in vitroADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S.Pat. No. 5,821,337 may be performed. Useful effector cells for suchassays include peripheral blood mononuclear cells (PBMC) and NaturalKiller (NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In certain embodiments, the FcR is a native sequencehuman FcR. Moreover, a preferred FcR is one that binds an IgG antibody(a gamma receptor) and includes receptors of the FcγRI, FcγRII, andFcγRIII subclasses, including allelic variants and alternatively splicedforms of these receptors. FCγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor FcγRIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (seereview M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs arereviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capelet al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126:330-41 (1995). Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein. The term alsoincludes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

“Human effector cells” are leukocytes that express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocytesthat mediate ADCC include PBMC, NK cells, monocytes, cytotoxic T cellsand neutrophils; with PBMCs and NK cells being preferred. The effectorcells may be isolated from a native source, e.g., from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)that are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed.

The terms “influenza A” and “Influenzavirus A” refer to a genus of theOrthomyxoviridae family of viruses. Influenzavirus A includes only onespecies: influenza A virus which cause influenza in birds, humans, pigs,and horses. Strains of all subtypes of influenza A virus have beenisolated from wild birds, although disease is uncommon. Some isolates ofinfluenza A virus cause severe disease both in domestic poultry and,rarely, in humans.

A “mammal” for purposes of treating n infection, refers to any mammal,including humans, domestic and farm animals, and zoo, sports, or petanimals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

“Treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures; wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisorder. Those in need of treatment include those already with thedisorder as well as those prone to have the disorder or those in whomthe disorder is to be prevented. A subject or mammal is successfully“treated” for an infection if, after receiving a therapeutic amount ofan antibody according to the methods of the present invention, thepatient shows observable and/or measurable reduction in or absence ofone or more of the following: reduction in the number of infected cellsor absence of the infected cells; reduction in the percent of totalcells that are infected; and/or relief to some extent, one or more ofthe symptoms associated with the specific infection; reduced morbidityand mortality, and improvement in quality of life issues. The aboveparameters for assessing successful treatment and improvement in thedisease are readily measurable by routine procedures familiar to aphysician.

The term “therapeutically effective amount” refers to an amount of anantibody or a drug effective to “treat” a disease or disorder in asubject or mammal. See preceding definition of “treating.”

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™ polyethylene glycol(PEG), and PLURONICS™.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹¹³, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g., methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, either in vitro or in vivo.Examples of growth inhibitory agents include agents that block cellcycle progression, such as agents that induce G1 arrest and M-phasearrest. Classical M-phase blockers include the vinca alkaloids(vincristine, vinorelbine and vinblastine), taxanes, and topoisomeraseII inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide,and bleomycin. Those agents that arrest G1 also spill over into S-phasearrest, for example, DNA alkylating agents such as tamoxifen,prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate,5-fluorouracil, and ara-C. Further information can be found in TheMolecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1,entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” byMurakami et al. (W B Saunders: Philadelphia, 1995), especially p. 13.The taxanes (paclitaxel and docetaxel) are anticancer drugs both derivedfrom the yew tree. Docetaxel (TAXOTERE™, Rhone-Poulenc Rorer), derivedfrom the European yew, is a semisynthetic analogue of paclitaxel(TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote theassembly of microtubules from tubulin dimers and stabilize microtubulesby preventing depolymerization, which results in the inhibition ofmitosis in cells.

“Label” as used herein refers to a detectable compound or compositionthat is conjugated directly or indirectly to the antibody so as togenerate a “labeled” antibody. The label may be detectable by itself(e.g., radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition that is detectable.

The term “epitope tagged” as used herein refers to a chimericpolypeptide comprising a polypeptide fused to a “tag polypeptide.” Thetag polypeptide has enough residues to provide an epitope against whichan antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide is also preferably fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to refer to single- or double-stranded RNA, DNA, or mixedpolymers. Polynucleotides may include genomic sequences, extra-genomicand plasmid sequences, and smaller engineered gene segments thatexpress, or may be adapted to express polypeptides.

An “isolated nucleic acid” is a nucleic acid that is substantiallyseparated from other genome DNA sequences as well as proteins orcomplexes such as ribosomes and polymerases, which naturally accompany anative sequence. The term embraces a nucleic acid sequence that has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analoguesor analogues biologically synthesized by heterologous systems. Asubstantially pure nucleic acid includes isolated forms of the nucleicacid. Of course, this refers to the nucleic acid as originally isolatedand does not exclude genes or sequences later added to the isolatednucleic acid by the hand of man.

The term “polypeptide” is used in its conventional meaning, i.e., as asequence of amino acids. The polypeptides are not limited to a specificlength of the product. Peptides, oligopeptides, and proteins areincluded within the definition of polypeptide, and such terms may beused interchangeably herein unless specifically indicated otherwise.This term also does not refer to or exclude post-expressionmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring. A polypeptide may be an entire protein, or asubsequence thereof. Particular polypeptides of interest in the contextof this invention are amino acid subsequences comprising CDRs and beingcapable of binding an antigen or Influenza A-infected cell.

An “isolated polypeptide” is one that has been identified and separatedand/or recovered from a component of its natural environment. Inpreferred embodiments, the isolated polypeptide will be purified (1) togreater than 95% by weight of polypeptide as determined by the Lowrymethod, and most preferably more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under reducing or non-reducing conditions usingCoomassie blue or, preferably, silver stain. Isolated polypeptideincludes the polypeptide in situ within recombinant cells since at leastone component of the polypeptide's natural environment will not bepresent. Ordinarily, however, isolated polypeptide will be prepared byat least one purification step.

A “native sequence” polynucleotide is one that has the same nucleotidesequence as a polynucleotide derived from nature. A “native sequence”polypeptide is one that has the same amino acid sequence as apolypeptide (e.g., antibody) derived from nature (e.g., from anyspecies). Such native sequence polynucleotides and polypeptides can beisolated from nature or can be produced by recombinant or syntheticmeans.

A polynucleotide “variant,” as the term is used herein, is apolynucleotide that typically differs from a polynucleotide specificallydisclosed herein in one or more substitutions, deletions, additionsand/or insertions. Such variants may be naturally occurring or may besynthetically generated, for example, by modifying one or more of thepolynucleotide sequences of the invention and evaluating one or morebiological activities of the encoded polypeptide as described hereinand/or using any of a number of techniques well known in the art.

A polypeptide “variant,” as the term is used herein, is a polypeptidethat typically differs from a polypeptide specifically disclosed hereinin one or more substitutions, deletions, additions and/or insertions.Such variants may be naturally occurring or may be syntheticallygenerated, for example, by modifying one or more of the abovepolypeptide sequences of the invention and evaluating one or morebiological activities of the polypeptide as described herein and/orusing any of a number of techniques well known in the art.

Modifications may be made in the structure of the polynucleotides andpolypeptides of the present invention and still obtain a functionalmolecule that encodes a variant or derivative polypeptide with desirablecharacteristics. When it is desired to alter the amino acid sequence ofa polypeptide to create an equivalent, or even an improved, variant orportion of a polypeptide of the invention, one skilled in the art willtypically change one or more of the codons of the encoding DNA sequence.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of its ability tobind other polypeptides (e.g., antigens) or cells. Since it is thebinding capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated that various changes may bemade in the peptide sequences of the disclosed compositions, orcorresponding DNA sequences that encode said peptides withoutappreciable loss of their biological utility or activity.

In many instances, a polypeptide variant will contain one or moreconservative substitutions. A “conservative substitution” is one inwhich an amino acid is substituted for another amino acid that hassimilar properties, such that one skilled in the art of peptidechemistry would expect the secondary structure and hydropathic nature ofthe polypeptide to be substantially unchanged.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like. Eachamino acid has been assigned a hydropathic index on the basis of itshydrophobicity and charge characteristics (Kyte and Doolittle, 1982).These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101states that the greatest local average hydrophilicity of a protein, asgoverned by the hydrophilicity of its adjacent amino acids, correlateswith a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Amino acid substitutions may further be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity and/orthe amphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine and valine; glycine and alanine; asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) ala, pro,gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Avariant may also, or alternatively, contain nonconservative changes. Ina preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of five amino acids orfewer. Variants may also (or alternatively) be modified by, for example,the deletion or addition of amino acids that have minimal influence onthe immunogenicity, secondary structure and hydropathic nature of thepolypeptide.

Polypeptides may comprise a signal (or leader) sequence at theN-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

When comparing polynucleotide and polypeptide sequences, two sequencesare said to be “identical” if the sequence of nucleotides or amino acidsin the two sequences is the same when aligned for maximumcorrespondence, as described below. Comparisons between two sequencesare typically performed by comparing the sequences over a comparisonwindow to identify and compare local regions of sequence similarity. A“comparison window” as used herein, refers to a segment of at leastabout 20 contiguous positions, usually 30 to about 75, 40 to about 50,in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides and polypeptides of the invention.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information.

In one illustrative example, cumulative scores can be calculated using,for nucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, and expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10,M=5, N=−4 and a comparison of both strands.

For amino acid sequences, a scoring matrix can be used to calculate thecumulative score. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment.

In one approach, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidues occur in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e., the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

“Homology” refers to the percentage of residues in the polynucleotide orpolypeptide sequence variant that are identical to the non-variantsequence after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology. In particularembodiments, polynucleotide and polypeptide variants have at least 70%,at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, orat least 99% polynucleotide or polypeptide homology with apolynucleotide or polypeptide described herein.

“Vector” includes shuttle and expression vectors. Typically, the plasmidconstruct will also include an origin of replication (e.g., the ColE1origin of replication) and a selectable marker (e.g., ampicillin ortetracycline resistance), for replication and selection, respectively,of the plasmids in bacteria. An “expression vector” refers to a vectorthat contains the necessary control sequences or regulatory elements forexpression of the antibodies including antibody fragment of theinvention, in bacterial or eukaryotic cells. Suitable vectors aredisclosed below.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

The present invention includes HuM2e antibodies comprising a polypeptideof the present invention, including those polypeptides encoded by apolynucleotide sequence set forth in Example 1 and amino acid sequencesset forth in Example 1 and 2, and fragments and variants thereof. In oneembodiment, the antibody is an antibody designated herein as 8i10,21B15, or 23K12. These antibodies preferentially bind to or specificallybind to influenza A infected cells as compared to uninfected controlcells of the same cell type.

In particular embodiments, the antibodies of the present invention bindto the M2 protein. In certain embodiments, the present inventionprovides HuM2e antibodies that bind to epitopes within M2e that are onlypresent in the native conformation, i.e., as expressed in cells. Inparticular embodiments, these antibodies fail to specifically bind to anisolated M2e polypeptide, e.g., the 23 amino acid residue M2e fragment.It is understood that these antibodies recognize non-linear (i.e.conformational) epitope(s) of the M2 peptide.

These specific conformational epitopes within the M2 protein, andparticularly within M2e, may be used as vaccines to prevent thedevelopment of influenza infection within a subject.

As will be understood by the skilled artisan, general description ofantibodies herein and methods of preparing and using the same also applyto individual antibody polypeptide constituents and antibody fragments.

The antibodies of the present invention may be polyclonal or monoclonalantibodies. However, in preferred embodiments, they are monoclonal. Inparticular embodiments, antibodies of the present invention are fullyhuman antibodies. Methods of producing polyclonal and monoclonalantibodies are known in the art and described generally, e.g., in U.S.Pat. No. 6,824,780. Typically, the antibodies of the present inventionare produced recombinantly, using vectors and methods available in theart, as described further below. Human antibodies may also be generatedby in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and5,229,275).

Human antibodies may also be produced in transgenic animals (e.g., mice)that are capable of producing a full repertoire of human antibodies inthe absence of endogenous immunoglobulin production. For example, it hasbeen described that the homozygous deletion of the antibody heavy-chainjoining region (J_(H)) gene in chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production.Transfer of the human germ-line immunoglobulin gene array into suchgerm-line mutant mice results in the production of human antibodies uponantigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci.USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);Bruggemann et al., Year in Immuno., 7:33 (1993); U.S. Pat. Nos.5,545,806, 5,569,825, 5,591,669 (all of GenPharm); U.S. Pat. No.5,545,807; and WO 97/17852. Such animals may be genetically engineeredto produce human antibodies comprising a polypeptide of the presentinvention.

In certain embodiments, antibodies of the present invention are chimericantibodies that comprise sequences derived from both human and non-humansources. In particular embodiments, these chimeric antibodies arehumanized or Primatized™. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

In the context of the present invention, chimeric antibodies alsoinclude fully human antibodies wherein the human hypervariable region orone or more CDRs are retained, but one or more other regions of sequencehave been replaced by corresponding sequences from a non-human animal.

The choice of non-human sequences, both light and heavy, to be used inmaking the chimeric antibodies is important to reduce antigenicity andhuman anti-non-human antibody responses when the antibody is intendedfor human therapeutic use. It is further important that chimericantibodies retain high binding affinity for the antigen and otherfavorable biological properties. To achieve this goal, according to apreferred method, chimeric antibodies are prepared by a process ofanalysis of the parental sequences and various conceptual chimericproducts using three-dimensional models of the parental human andnon-human sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the recipient andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the hypervariable region residues are directly and most substantiallyinvolved in influencing antigen binding.

As noted above, antibodies (or immunoglobulins) can be divided into fivedifferent classes, based on differences in the amino acid sequences inthe constant region of the heavy chains. All immunoglobulins within agiven class have very similar heavy chain constant regions. Thesedifferences can be detected by sequence studies or more commonly byserological means (i.e. by the use of antibodies directed to thesedifferences). Antibodies, or fragments thereof, of the present inventionmay be any class, and may, therefore, have a gamma, mu, alpha, delta, orepsilon heavy chain. A gamma chain may be gamma 1, gamma 2, gamma 3, orgamma 4; and an alpha chain may be alpha 1 or alpha 2.

In a preferred embodiment, an antibody of the present invention, orfragment thereof, is an IgG. IgG is considered the most versatileimmunoglobulin, because it is capable of carrying out all of thefunctions of immunoglobulin molecules. IgG is the major Ig in serum, andthe only class of Ig that crosses the placenta. IgG also fixescomplement, although the IgG4 subclass does not. Macrophages, monocytes,PMN's and some lymphocytes have Fc receptors for the Fc region of IgG.Not all subclasses bind equally well; IgG2 and IgG4 do not bind to Fcreceptors. A consequence of binding to the Fc receptors on PMN's,monocytes and macrophages is that the cell can now internalize theantigen better. IgG is an opsonin that enhances phagocytosis. Binding ofIgG to Fc receptors on other types of cells results in the activation ofother functions. Antibodies of the present invention may be of any IgGsubclass.

In another preferred embodiment, an antibody, or fragment thereof, ofthe present invention is an IgE. IgE is the least common serum Ig sinceit binds very tightly to Fc receptors on basophils and mast cells evenbefore interacting with antigen. As a consequence of its binding tobasophils an mast cells, IgE is involved in allergic reactions. Bindingof the allergen to the IgE on the cells results in the release ofvarious pharmacological mediators that result in allergic symptoms. IgEalso plays a role in parasitic helminth diseases. Eosinophils have Fcreceptors for IgE and binding of eosinophils to IgE-coated helminthsresults in killing of the parasite. IgE does not fix complement.

In various embodiments, antibodies of the present invention, andfragments thereof, comprise a variable light chain that is either kappaor lambda. The lamba chain may be any of subtype, including, e.g.,lambda 1, lambda 2, lambda 3, and lambda 4.

As noted above, the present invention further provides antibodyfragments comprising a polypeptide of the present invention. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. For example, the smaller size of the fragmentsallows for rapid clearance, and may lead to improved access to certaintissues, such as solid tumors. Examples of antibody fragments include:Fab, Fab′, F(ab′)₂ and Fv fragments; diabodies; linear antibodies;single-chain antibodies; and multispecific antibodies formed fromantibody fragments.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Fab′-SH fragments can be directly recovered from E. coli and chemicallycoupled to form F(ab′)₂ fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another approach, F(ab′)₂ fragments can beisolated directly from recombinant host cell culture. Fab and F(ab′)₂fragment with increased in vivo half-life comprising a salvage receptorbinding epitope residues are described in U.S. Pat. No. 5,869,046. Othertechniques for the production of antibody fragments will be apparent tothe skilled practitioner.

In other embodiments, the antibody of choice is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and sFv are the only species with intact combining sitesthat are devoid of constant regions. Thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins may beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

In certain embodiments, antibodies of the present invention arebispecific or multi-specific. Bispecific antibodies are antibodies thathave binding specificities for at least two different epitopes.Exemplary bispecific antibodies may bind to two different epitopes of asingle antigen. Other such antibodies may combine a first antigenbinding site with a binding site for a second antigen. Alternatively, ananti-M2e arm may be combined with an arm that binds to a triggeringmolecule on a leukocyte, such as a T-cell receptor molecule (e.g., CD3),or Fc receptors for IgG (FcγR), such as FcγRIl (CD64), FcγRII (CD32) andFcγRIII (CD16), so as to focus and localize cellular defense mechanismsto the infected cell. Bispecific antibodies may also be used to localizecytotoxic agents to infected cells. These antibodies possess anM2e-binding arm and an arm that binds the cytotoxic agent (e.g.,saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexateor radioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g., F(ab′)₂ bispecificantibodies). WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIIIantibody and U.S. Pat. No. 5,837,234 discloses a bispecificanti-ErbB2/anti-FcγRI antibody. A bispecific anti-ErbB2/Fcα antibody isshown in WO98/02463. U.S. Pat. No. 5,821,337 teaches a bispecificanti-ErbB2/anti-CD3 antibody.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. Preferably, thefusion is with an Ig heavy chain constant domain, comprising at leastpart of the hinge, C_(H)2, and C_(H)3 regions. It is preferred to havethe first heavy-chain constant region (C_(H)1) containing the sitenecessary for light chain bonding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable host cell.This provides for greater flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yield of the desired bispecific antibody. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains into a single expression vector when the expressionof at least two polypeptide chains in equal ratios results in highyields or when the ratios have no significant affect on the yield of thedesired chain combination.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers that are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g., alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agent,sodium arsenite, to stabilize vicinal dithiols and preventintermolecular disulfide formation. The Fab′ fragments generated arethen converted to thionitrobenzoate (TNB) derivatives. One of theFab′-TNB derivatives is then reconverted to the Fab′-thiol by reductionwith mercaptoethylamine and is mixed with an equimolar amount of theother Fab′-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for the selectiveimmobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise a V_(H)connected to a V_(L) by a linker that is too short to allow pairingbetween the two domains on the same chain. Accordingly, the V_(H) andV_(L) domains of one fragment are forced to pair with the complementaryV_(L) and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60(1991). A multivalent antibody may be internalized (and/or catabolized)faster than a bivalent antibody by a cell expressing an antigen to whichthe antibodies bind. The antibodies of the present invention can bemultivalent antibodies with three or more antigen binding sites (e.g.,tetravalent antibodies), which can be readily produced by recombinantexpression of nucleic acid encoding the polypeptide chains of theantibody. The multivalent antibody can comprise a dimerization domainand three or more antigen binding sites. The preferred dimerizationdomain comprises (or consists of) an Fc region or a hinge region. Inthis scenario, the antibody will comprise an Fc region and three or moreantigen binding sites amino-terminal to the Fc region. The preferredmultivalent antibody herein comprises (or consists of) three to abouteight, but preferably four, antigen binding sites. The multivalentantibody comprises at least one polypeptide chain (and preferably twopolypeptide chains), wherein the polypeptide chain(s) comprise two ormore variable domains. For instance, the polypeptide chain(s) maycomprise VD1-(X1)_(n)-VD2-(X2)_(n)-Fc, wherein VD1 is a first variabledomain, VD2 is a second variable domain, Fc is one polypeptide chain ofan Fc region, X1 and X2 represent an amino acid or polypeptide, and n is0 or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein preferably furthercomprises at least two (and preferably four) light chain variable domainpolypeptides. The multivalent antibody herein may, for instance,comprise from about two to about eight light chain variable domainpolypeptides. The light chain variable domain polypeptides contemplatedhere comprise a light chain variable domain and, optionally, furthercomprise a C_(L) domain.

Antibodies of the present invention further include single chainantibodies.

In particular embodiments, antibodies of the present invention areinternalizing antibodies.

Amino acid sequence modification(s) of the antibodies described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody.Amino acid sequence variants of the antibody may be prepared byintroducing appropriate nucleotide changes into a polynucleotide thatencodes the antibody, or a chain thereof, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution may be made to arrive at the final antibody, provided thatthe final construct possesses the desired characteristics. The aminoacid changes also may alter post-translational processes of theantibody, such as changing the number or position of glycosylationsites. Any of the variations and modifications described above forpolypeptides of the present invention may be included in antibodies ofthe present invention.

A useful method for identification of certain residues or regions of anantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells in Science,244:1081-1085 (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with PSCA antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed anti-antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of an antibodyinclude the fusion to the N- or C-terminus of the antibody to an enzyme(e.g., for ADEPT) or a polypeptide that increases the serum half-life ofthe antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative and non-conservativesubstitutions are contemplated.

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody. Generally, theresulting variant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g., 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g., binding affinity) asherein disclosed. In order to identify candidate hypervariable regionsites for modification, alanine scanning mutagenesis can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and an antigen or infected cell.Such contact residues and neighboring residues are candidates forsubstitution according to the techniques elaborated herein. Once suchvariants are generated, the panel of variants is subjected to screeningas described herein and antibodies with superior properties in one ormore relevant assays may be selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

The antibody of the invention is modified with respect to effectorfunction, e.g., so as to enhance antigen-dependent cell-mediatedcyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of theantibody. This may be achieved by introducing one or more amino acidsubstitutions in an Fc region of the antibody. Alternatively oradditionally, cysteine residue(s) may be introduced in the Fc region,thereby allowing interchain disulfide bond formation in this region. Thehomodimeric antibody thus generated may have improved internalizationcapability and/or increased complement-mediated cell killing andantibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J.Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922(1992). Homodimeric antibodies with enhanced anti-infection activity mayalso be prepared using heterobifunctional cross-linkers as described inWolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, anantibody can be engineered which has dual Fc regions and may therebyhave enhanced complement lysis and ADCC capabilities. See Stevenson etal., Anti-Cancer Drug Design 3:219-230 (1989).

To increase the serum half-life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Antibodies of the present invention may also be modified to include anepitope tag or label, e.g., for use in purification or diagnosticapplications. The invention also pertains to therapy withimmunoconjugates comprising an antibody conjugated to an anti-canceragent such as a cytotoxic agent or a growth inhibitory agent.Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, a trichothene, and CC1065, and thederivatives of these toxins that have toxin activity, are alsocontemplated herein.

In one preferred embodiment, an antibody (full length or fragments) ofthe invention is conjugated to one or more maytansinoid molecules.Maytansinoids are mitototic inhibitors that act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

In an attempt to improve their therapeutic index, maytansine andmaytansinoids have been conjugated to antibodies specifically binding totumor cell antigens. Immunoconjugates containing maytansinoids and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1. Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprisinga maytansinoid designated DM1 linked to the monoclonal antibody C242directed against human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. An average of 3-4 maytansinoid molecules conjugated perantibody molecule has shown efficacy in enhancing cytotoxicity of targetcells without negatively affecting the function or solubility of theantibody, although even one molecule of toxin/antibody would be expectedto enhance cytotoxicity over the use of naked antibody. Maytansinoidsare well known in the art and can be synthesized by known techniques orisolated from natural sources. Suitable maytansinoids are disclosed, forexample, in U.S. Pat. No. 5,208,020 and in the other patents andnonpatent publications referred to hereinabove. Preferred maytansinoidsare maytansinol and maytansinol analogues modified in the aromatic ringor at other positions of the maytansinol molecule, such as variousmaytansinol esters.

There are many linking groups known in the art for making antibodyconjugates, including, for example, those disclosed in U.S. Pat. No.5,208,020 or EP Patent 0 425 235 B1, and Chari et al., Cancer Research52: 127-131 (1992). The linking groups include disufide groups,thioether groups, acid labile groups, photolabile groups, peptidaselabile groups, or esterase labile groups, as disclosed in theabove-identified patents, disulfide and thioether groups beingpreferred.

Immunoconjugates may be made using a variety of bifunctional proteincoupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate(SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson etal., Biochem. J. 173:723-737 [1978]) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage. For example, a ricin immunotoxin can be prepared asdescribed in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody. See WO94/11026. The linker may be a“cleavable linker” facilitating release of the cytotoxic drug in thecell. For example, an acid-labile linker, Cancer Research 52: 127-131(1992); U.S. Pat. No. 5,208,020) may be used.

Another immunoconjugate of interest comprises an antibody conjugated toone or more calicheamicin molecules. The calicheamicin family ofantibiotics are capable of producing double-stranded DNA breaks atsub-picomolar concentrations. For the preparation of conjugates of thecalicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586,5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all toAmerican Cyanamid Company). Another drug that the antibody can beconjugated is QFA which is an antifolate. Both calicheamicin and QFAhave intracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Examples of other agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof that can be usedinclude, e.g., diphtheria A chain, nonbinding active fragments ofdiphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricinA chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232.

The present invention further includes an immunoconjugate formed betweenan antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of infected cells, the antibody includes ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated anti-PSCA antibodies. Examplesinclude At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Rc¹⁸⁸, Sm¹⁵³, Bi²¹², P³², P²¹²and radioactive isotopes of Lu. When the conjugate is used fordiagnosis, it may comprise a radioactive atom for scintigraphic studies,for example tc^(99m) or I¹²³, or a spin label for nuclear magneticresonance (NMR) imaging (also known as magnetic resonance imaging, mri),such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other label is incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al. (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent is made, e.g., by recombinant techniques or peptide synthesis. Thelength of DNA may comprise respective regions encoding the two portionsof the conjugate either adjacent one another or separated by a regionencoding a linker peptide which does not destroy the desired propertiesof the conjugate.

The antibodies of the present invention are also used in antibodydependent enzyme mediated prodrug therapy (ADET) by conjugating theantibody to a prodrug-activating enzyme which converts a prodrug (e.g.,a peptidyl chemotherapeutic agent, see WO81/01145) to an activeanti-cancer drug (see, e.g., WO 88/07378 and U.S. Pat. No. 4,975,278).

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form. Enzymes that are useful in themethod of this invention include, but are not limited to, alkalinephosphatase useful for converting phosphate-containing prodrugs intofree drugs; arylsulfatase useful for converting sulfate-containingprodrugs into free drugs; cytosine deaminase useful for convertingnon-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;proteases, such as serratia protease, thermolysin, subtilisin,carboxypeptidases and cathepsins (such as cathepsins B and L), that areuseful for converting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, useful for converting prodrugs that containD-amino acid substituents; carbohydrate-cleaving enzymes such asβ-galactosidase and neuraminidase useful for converting glycosylatedprodrugs into free drugs; β-lactamase useful for converting drugsderivatized with β-lactams into free drugs; and penicillin amidases,such as penicillin V amidase or penicillin G amidase, useful forconverting drugs derivatized at their amine nitrogens with phenoxyacetylor phenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs of the invention into free activedrugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme to a infected cell population.

The enzymes of this invention can be covalently bound to the antibodiesby techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 312: 604-608 (1984).

Other modifications of the antibody are contemplated herein. Forexample, the antibody may be linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol. The antibody also may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate)microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,(1980).

The antibodies disclosed herein are also formulated as immunoliposomes.A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant that is useful for delivery of a drug toa mammal. The components of the liposome are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes. Liposomes containing the antibody are prepared by methodsknown in the art, such as described in Epstein et al., Proc. Natl. Acad.Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA,77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731published Oct. 23, 1997. Liposomes with enhanced circulation time aredisclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desired adiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al., J. National Cancer Inst. 81(19)1484 (1989).

Antibodies of the present invention, or fragments thereof may possessany of a variety of biological or functional characteristics. In certainembodiments, these antibodies are Influenza A specific or M2 proteinspecific antibodies, indicating that they specifically bind to orpreferentially bind to Influenza A or the M2 protein thereof,respectively, as compared to a normal control cell. In particularembodiments, the antibodies are HuM2e antibodies, indicating that theyspecifically bind to a M2e protein, preferably to an epitope of the M2edomain that is only present when the M2 protein is expressed in cells orpresent on a virus, as compared to a normal control cell.

In particular embodiments, an antibody of the present invention is anantagonist antibody, which partially or fully blocks or inhibits abiological activity of a polypeptide or cell to which it specifically orpreferentially binds. In other embodiments, an antibody of the presentinvention is a growth inhibitory antibody, which partially or fullyblocks or inhibits the growth of an infected cell to which it binds. Inanother embodiment, an antibody of the present invention inducesapoptosis. In yet another embodiment, an antibody of the presentinvention induces or promotes antibody-dependent cell-mediatedcytotoxicity or complement dependent cytotoxicity.

Methods of Identifying and Producing Antibodies Specific for InfluenzaVirus

The present invention provides novel methods for the identification ofHuM2e antibodies, as exemplified in Example 4. These methods may bereadily adapted to identify antibodies specific for other polypeptidesexpressed on the cell surface by infectious agents, or even polypeptidesexpressed on the surface of an infectious agent itself.

In general, the methods include obtaining serum samples from patientsthat have been infected with or vaccinated against an infectious agent.These serum samples are then screened to identify those that containantibodies specific for a particular polypeptide associated with theinfectious agent, such as, e.g., a polypeptide specifically expressed onthe surface of cells infected with the infectious agent, but notuninfected cells. In particular embodiments, the serum samples arescreened by contacting the samples with a cell that has been transfectedwith an expression vector that expresses the polypeptide expressed onthe surface of infected cells.

Once a patient is identified as having serum containing an antibodyspecific for the infectious agent polypeptide of interest is identified,mononuclear and/or B cells obtained from the same patient are used toidentify a cell or clone thereof that produces the antibody, using anyof the methods described herein or available in the art. Once a B cellthat produces the antibody is identified, cDNAs encoding the variableregions or fragments thereof of the antibody may be cloned usingstandard RT-PCR vectors and primers specific for conserved antibodysequences, and subcloned in to expression vectors used for therecombinant production of monoclonal antibodies specific for theinfectious agent polypeptide of interest.

In one embodiment, the present invention provides a method ofidentifying an antibody that specifically binds influenza A-infectedcells, comprising: contacting an Influenza A virus or a cell expressingthe M2 protein with a biological sample obtained from a patient havingbeen infected by Influenza A; determining an amount of antibody in thebiological sample that binds to the cell; and comparing the amountdetermined with a control value, wherein if the value determined is atleast two-fold greater than the control value, an antibody thatspecifically binds influenza A-infected cells is indicated. In variousembodiments, the cells expressing an M2 protein are cells infected withan Influenza A virus or cells that have been transfected with apolynucleotide that expressed the M2 protein. Alternatively, the cellsmay express a portion of the M2 protein that includes the M2e domain andenough additional M2 sequence that the protein remains associated withthe cell and the M2e domain is presented on the cell surface in the samemanner as when present within full length M2 protein. Methods ofpreparing an M2 expression vector and transfecting an appropriate cell,including those described herein, may be readily accomplished, in viewof the M2 sequence being publicly available. See, for example, theInfluenza Sequence Database (ISD) (flu.lan1.gov on the World Wide Web,described in Macken et al., 2001, “The value of a database insurveillance and vaccine selection” in Options for the Control ofInfluenza IV. A.D.M.E., Osterhaus & Hampson (Eds.), Elsevier Science,Amsterdam, pp. 103-106) and the Microbial Sequencing Center (MSC) at TheInstitute for Genomic Research (TIGR) (tigr.org/msc/infl_a_virus.shtmlon the World Wide Web).

The M2e-expressing cells or virus described above are used to screen thebiological sample obtained from a patient infected with influenza A forthe presence of antibodies that preferentially bind to the cellexpressing the M2 polypeptide using standard biological techniques. Forexample, in certain embodiments, the antibodies may be labeled, and thepresence of label associated with the cell detected, e.g., using FMAT orFACs analysis. In particular embodiments, the biological sample isblood, serum, plasma, bronchial lavage, or saliva. Methods of thepresent invention may be practiced using high throughput techniques.

Identified human antibodies may then be characterized further. Forexample the particular conformational epitopes with in the M2e proteinthat are necessary or sufficient for binding of the antibody may bedetermined, e.g., using site-directed mutagenesis of expressed M2epolypeptides. These methods may be readily adapted to identify humanantibodies that bind any protein expressed on a cell surface.Furthermore, these methods may be adapted to determine binding of theantibody to the virus itself, as opposed to a cell expressingrecombinant M2e or infected with the virus.

Polynucleotide sequences encoding the antibodies, variable regionsthereof, or antigen-binding fragments thereof may be subcloned intoexpression vectors for the recombinant production of HuM2e antibodies.In one embodiment, this is accomplished by obtaining mononuclear cellsfrom the patient from the serum containing the identified HuM2e antibodywas obtained; producing B cell clones from the mononuclear cells;inducing the B cells to become antibody-producing plasma cells; andscreening the supernatants produced by the plasma cells to determine ifit contains the HuM2e antibody. Once a B cell clone that produces anHuM2e antibody is identified, reverse-transcription polymerase chainreaction (RT-PCR) is performed to clone the DNAs encoding the variableregions or portions thereof of the HuM2e antibody. These sequences arethen subcloned into expression vectors suitable for the recombinantproduction of human HuM2e antibodies. The binding specificity may beconfirmed by determining the recombinant antibody's ability to bindcells expressing M2e polypeptide.

In particular embodiments of the methods described herein, B cellsisolated from peripheral blood or lymph nodes are sorted, e.g., based ontheir being CD19 positive, and plated, e.g., as low as a single cellspecificity per well, e.g., in 96, 384, or 1536 well configurations. Thecells are induced to differentiate into antibody-producing cells, e.g.,plasma cells, and the culture supernatants are harvested and tested forbinding to cells expressing the infectious agent polypeptide on theirsurface using, e.g., FMAT or FACS analysis. Positive wells are thensubjected to whole well RT-PCR to amplify heavy and light chain variableregions of the IgG molecule expressed by the clonal daughter plasmacells. The resulting PCR products encoding the heavy and light chainvariable regions, or portions thereof, are subcloned into human antibodyexpression vectors for recombinant expression. The resulting recombinantantibodies are then tested to confirm their original binding specificityand may be further tested for pan-specificity across various strains ofisolates of the infectious agent.

Thus, in one embodiment, a method of identifying HuM2e antibodies ispracticed as follows. First, full length or approximately full length M2cDNAs are transfected into a cell line for expression of M2 protein.Secondly, individual human plasma or sera samples are tested forantibodies that bind the cell-expressed M2. And lastly, MAbs derivedfrom plasma- or serum-positive individuals are characterized for bindingto the same cell-expressed M2. Further definition of the finespecificities of the MAbs can be performed at this point.

These methods may be practiced to identify a variety of different HuM2eantibodies, including antibodies specific for (a) epitopes in a linearM2e peptide, (b) common epitopes in multiple variants of M2e, (c)conformational determinants of an M2 homotetramer, and (d) commonconformational determinants of multiple variants of the M2 homotetramer.The last category is particularly desirable, as this specificity isperhaps specific for all A strains of influenza.

Polynucleotides that encode the HuM2e antibodies or portions thereof ofthe present invention may be isolated from cells expressing HuM2eantibodies, according to methods available in the art and describedherein, including amplification by polymerase chain reaction usingprimers specific for conserved regions of human antibody polypeptides.For example, light chain and heavy chain variable regions may be clonedfrom the B cell according to molecular biology techniques described inWO 92/02551; U.S. Pat. No. 5,627,052; or Babcook et al., Proc. Natl.Acad. Sci. USA 93:7843-48 (1996). In certain embodiments,polynucleotides encoding all or a region of both the heavy and lightchain variable regions of the IgG molecule expressed by the clonaldaughter plasma cells expressing the HuM2e antibody are subcloned andsequenced. The sequence of the encoded polypeptide may be readilydetermined from the polynucleotide sequence. Isolated polynucleotidesencoding a polypeptide of the present invention may be subcloned into anexpression vector to recombinantly produce antibodies and polypeptidesof the present invention, using procedures known in the art anddescribed herein.

Binding properties of an antibody (or fragment thereof) to M2e orinfected cells or tissues may generally be determined and assessed usingimmunodetection methods including, for example, immunofluorescence-basedassays, such as immuno-histochemistry (IHC) and/orfluorescence-activated cell sorting (FACS). Immunoassay methods mayinclude controls and procedures to determine whether antibodies bindspecifically to M2e from one or more specific strains of Influenza A,and do not recognize or cross-react with normal control cells.

Following pre-screening of serum to identify patients that produceantibodies to an infectious agent or polypeptide thereof, e.g., M2, themethods of the present invention typically include the isolation orpurification of B cells from a biological sample previously obtainedfrom a patient or subject. The patient or subject may be currently orpreviously diagnosed with or suspect or having a particular disease orinfection, or the patient or subject may be considered free or aparticular disease or infection. Typically, the patient or subject is amammal and, in particular embodiments, a human. The biological samplemay be any sample that contains B cells, including but not limited to,lymph node or lymph node tissue, pleural effusions, peripheral blood,ascites, tumor tissue, or cerebrospinal fluid (CSF). In variousembodiments, B cells are isolated from different types of biologicalsamples, such as a biological sample affected by a particular disease orinfection. However, it is understood that any biological samplecomprising B cells may be used for any of the embodiments of the presentinvention.

Once isolated, the B cells are induced to produce antibodies, e.g., byculturing the B cells under conditions that support B cell proliferationor development into a plasmacyte, plasmablast, or plasma cell. Theantibodies are then screened, typically using high throughputtechniques, to identify an antibody that specifically binds to a targetantigen, e.g., a particular tissue, cell, infectious agent, orpolypeptide. In certain embodiments, the specific antigen, e.g., cellsurface polypeptide bound by the antibody is not known, while in otherembodiments, the antigen specifically bound by the antibody is known.

According to the present invention, B cells may be isolated from abiological sample, e.g., a tumor, tissue, peripheral blood or lymph nodesample, by any means known and available in the art. B cells aretypically sorted by FACS based on the presence on their surface of a Bcell-specific marker, e.g., CD19, CD138, and/or surface IgG. However,other methods known in the art may be employed, such as, e.g., columnpurification using CD19 magnetic beads or IgG-specific magnetic beads,followed by elution from the column. However, magnetic isolation of Bcells utilizing any marker may result in loss of certain B cells.Therefore, in certain embodiments, the isolated cells are not sortedbut, instead, phicol-purified mononuclear cells isolated from tumor aredirectly plated to the appropriate or desired number of specificitiesper well.

In order to identify B cells that produce an infectious agent-specificantibody, the B cells are typically plated at low density (e.g., asingle cell specificity per well, 1-10 cells per well, 10-100 cells perwell, 1-100 cells per well, less than 10 cells per well, or less than100 cells per well) in multi-well or microtitre plates, e.g., in 96,384, or 1536 well configurations. When the B cells are initially platedat a density greater than one cell per well, then the methods of thepresent invention may include the step of subsequently diluting cells ina well identified as producing an antigen-specific antibody, until asingle cell specificity per well is achieved, thereby facilitating theidentification of the B cell that produces the antigen-specificantibody. Cell supernatants or a portion thereof and/or cells may befrozen and stored for future testing and later recovery of antibodypolynucleotides.

In certain embodiments, the B cells are cultured under conditions thatfavor the production of antibodies by the B cells. For example, the Bcells may be cultured under conditions favorable for B cellproliferation and differentiation to yield antibody-producingplasmablast, plasmacytes, or plasma cells. In particular embodiments,the B cells are cultured in the presence of a B cell mitogen, such aslipopolysaccharide (LPS) or CD40 ligand. In one specific embodiment, Bcells are differentiated to antibody-producing cells by culturing themwith feed cells and/or other B cell activators, such as CD40 ligand.

Cell culture supernatants or antibodies obtained therefrom may be testedfor their ability to bind to a target antigen, using routine methodsavailable in the art, including those described herein. In particularembodiments, culture supernatants are tested for the presence ofantibodies that bind to a target antigen using high-throughput methods.For example, B cells may be cultured in multi-well microtitre dishes,such that robotic plate handlers may be used to simultaneously samplemultiple cell supernatants and test for the presence of antibodies thatbind to a target antigen. In particular embodiments, antigens are boundto beads, e.g., paramagnetic or latex beads) to facilitate the captureof antibody/antigen complexes. In other embodiments, antigens andantibodies are fluorescently labeled (with different labels) and FACSanalysis is performed to identify the presence of antibodies that bindto target antigen. In one embodiment, antibody binding is determinedusing FMAT™ analysis and instrumentation (Applied Biosystems, FosterCity, Calif.). FMAT™ is a fluorescence macro-confocal platform forhigh-throughput screening, which mix-and-read, non-radioactive assaysusing live cells or beads.

In the context of comparing the binding of an antibody to a particulartarget antigen (e.g., a biological sample such as infected tissue orcells, or infectious agents) as compared to a control sample (e.g., abiological sample such as uninfected cells, or a different infectiousagent), in various embodiments, the antibody is considered topreferentially bind a particular target antigen if at least two-fold, atleast three-fold, at least five-fold, or at least ten-fold more antibodybinds to the particular target antigen as compared to the amount thatbinds a control sample.

Polynucleotides encoding antibody chains, variable regions thereof, orfragments thereof, may be isolated from cells utilizing any meansavailable in the art. In one embodiment, polynucleotides are isolatedusing polymerase chain reaction (PCR), e.g., reverse transcription-PCR(RT-PCR) using oligonucleotide primers that specifically bind to heavyor light chain encoding polynucleotide sequences or complements thereofusing routine procedures available in the art. In one embodiment,positive wells are subjected to whole well RT-PCR to amplify the heavyand light chain variable regions of the IgG molecule expressed by theclonal daughter plasma cells. These PCR products may be sequenced.

The resulting PCR products encoding the heavy and light chain variableregions or portions thereof are then subcloned into human antibodyexpression vectors and recombinantly expressed according to routineprocedures in the art (see, e.g., U.S. Pat. No. 7,112,439). The nucleicacid molecules encoding a tumor-specific antibody or fragment thereof,as described herein, may be propagated and expressed according to any ofa variety of well-known procedures for nucleic acid excision, ligation,transformation, and transfection. Thus, in certain embodimentsexpression of an antibody fragment may be preferred in a prokaryotichost cell, such as Escherichia coli (see, e.g., Pluckthun et al.,Methods Enzymol. 178:497-515 (1989)). In certain other embodiments,expression of the antibody or an antigen-binding fragment thereof may bepreferred in a eukaryotic host cell, including yeast (e.g.,Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichiapastoris); animal cells (including mammalian cells); or plant cells.Examples of suitable animal cells include, but are not limited to,myeloma, COS, CHO, or hybridoma cells. Examples of plant cells includetobacco, corn, soybean, and rice cells. By methods known to those havingordinary skill in the art and based on the present disclosure, a nucleicacid vector may be designed for expressing foreign sequences in aparticular host system, and then polynucleotide sequences encoding thetumor-specific antibody (or fragment thereof) may be inserted. Theregulatory elements will vary according to the particular host.

One or more replicable expression vectors containing a polynucleotideencoding a variable and/or constant region may be prepared and used totransform an appropriate cell line, for example, a non-producing myelomacell line, such as a mouse NSO line or a bacteria, such as E. coli, inwhich production of the antibody will occur. In order to obtainefficient transcription and translation, the polynucleotide sequence ineach vector should include appropriate regulatory sequences,particularly a promoter and leader sequence operatively linked to thevariable domain sequence. Particular methods for producing antibodies inthis way are generally well known and routinely used. For example,molecular biology procedures are described by Sambrook et al. (MolecularCloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory,New York, 1989; see also Sambrook et al., 3rd ed., Cold Spring HarborLaboratory, New York, (2001)). While not required, in certainembodiments, regions of polynucleotides encoding the recombinantantibodies may be sequenced. DNA sequencing can be performed asdescribed in Sanger et al. (Proc. Natl. Acad. Sci. USA 74:5463 (1977))and the Amersham International plc sequencing handbook and includingimprovements thereto.

In particular embodiments, the resulting recombinant antibodies orfragments thereof are then tested to confirm their original specificityand may be further tested for pan-specificity, e.g., with relatedinfectious agents. In particular embodiments, an antibody identified orproduced according to methods described herein is tested for cellkilling via antibody dependent cellular cytotoxicity (ADCC) orapoptosis, and/or well as its ability to internalize.

Polynucleotides

The present invention, in other aspects, provides polynucleotidecompositions. In preferred embodiments, these polynucleotides encode apolypeptide of the invention, e.g., a region of a variable chain of anantibody that binds to Influenza A, M2, or M2e. Polynucleotides of theinvention are single-stranded (coding or antisense) or double-strandedDNA (genomic, cDNA or synthetic) or RNA molecules. RNA moleculesinclude, but are not limited to, HnRNA molecules, which contain intronsand correspond to a DNA molecule in a one-to-one manner, and mRNAmolecules, which do not contain introns. Alternatively, or in addition,coding or non-coding sequences are present within a polynucleotide ofthe present invention. Also alternatively, or in addition, apolynucleotide is linked to other molecules and/or support materials ofthe invention. Polynucleotides of the invention are used, e.g., inhybridization assays to detect the presence of an Influenza A antibodyin a biological sample, and in the recombinant production ofpolypeptides of the invention.

Therefore, according to another aspect of the present invention,polynucleotide compositions are provided that include some or all of apolynucleotide sequence set forth in Example 1, complements of apolynucleotide sequence set forth in Example 1, and degenerate variantsof a polynucleotide sequence set forth in Example 1. In certainpreferred embodiments, the polynucleotide sequences set forth hereinencode polypeptides capable of preferentially binding a InfluenzaA-infected cell as compared to a normal control uninfected cell,including a polypeptide having a sequence set forth in Examples 1 or 2.Furthermore, the invention includes all polynucleotides that encode anypolypeptide of the present invention.

In other related embodiments, the invention provides polynucleotidevariants having substantial identity to the sequences set forth in FIG.1, for example those comprising at least 70% sequence identity,preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% orhigher, sequence identity compared to a polynucleotide sequence of thisinvention, as determined using the methods described herein, (e.g.,BLAST analysis using standard parameters). One skilled in this art willrecognize that these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning, and the like.

Typically, polynucleotide variants contain one or more substitutions,additions, deletions and/or insertions, preferably such that theimmunogenic binding properties of the polypeptide encoded by the variantpolynucleotide is not substantially diminished relative to a polypeptideencoded by a polynucleotide sequence specifically set forth herein. Inadditional embodiments, the present invention provides polynucleotidefragments comprising various lengths of contiguous stretches of sequenceidentical to or complementary to one or more of the sequences disclosedherein. For example, polynucleotides are provided by this invention thatcomprise at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300,400, 500 or 1000 or more contiguous nucleotides of one or more of thesequences disclosed herein as well as all intermediate lengths therebetween. As used herein, the term “intermediate lengths” is meant todescribe any length between the quoted values, such as 16, 17, 18, 19,etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100,101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integersthrough 200-500; 500-1,000, and the like.

In another embodiment of the invention, polynucleotide compositions areprovided that are capable of hybridizing under moderate to highstringency conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridizing at 50° C.-60° C., 5×SSC, overnight;followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5×and 0.2×SSC containing 0.1% SDS. One skilled in the art will understandthat the stringency of hybridization can be readily manipulated, such asby altering the salt content of the hybridization solution and/or thetemperature at which the hybridization is performed. For example, inanother embodiment, suitable highly stringent hybridization conditionsinclude those described above, with the exception that the temperatureof hybridization is increased, e.g., to 60-65° C. or 65-70° C.

In preferred embodiments, the polypeptide encoded by the polynucleotidevariant or fragment has the same binding specificity (i.e., specificallyor preferentially binds to the same epitope or Influenza A strain) asthe polypeptide encoded by the native polynucleotide. In certainpreferred embodiments, the polynucleotides described above, e.g.,polynucleotide variants, fragments and hybridizing sequences, encodepolypeptides that have a level of binding activity of at least about50%, preferably at least about 70%, and more preferably at least about90% of that for a polypeptide sequence specifically set forth herein.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. A nucleic acid fragment of almost any length is employed,with the total length preferably being limited by the ease ofpreparation and use in the intended recombinant DNA protocol. Forexample, illustrative polynucleotide segments with total lengths ofabout 10,000, about 5000, about 3000, about 2,000, about 1,000, about500, about 200, about 100, about 50 base pairs in length, and the like,(including all intermediate lengths) are included in manyimplementations of this invention.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are multiplenucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that encode apolypeptide of the present invention but which vary due to differencesin codon usage are specifically contemplated by the invention. Further,alleles of the genes including the polynucleotide sequences providedherein are within the scope of the invention. Alleles are endogenousgenes that are altered as a result of one or more mutations, such asdeletions, additions and/or substitutions of nucleotides. The resultingmRNA and protein may, but need not, have an altered structure orfunction. Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

In certain embodiments of the present invention, mutagenesis of thedisclosed polynucleotide sequences is performed in order to alter one ormore properties of the encoded polypeptide, such as its bindingspecificity or binding strength. Techniques for mutagenesis arewell-known in the art, and are widely used to create variants of bothpolypeptides and polynucleotides. A mutagenesis approach, such assite-specific mutagenesis, is employed for the preparation of variantsand/or derivatives of the polypeptides described herein. By thisapproach, specific modifications in a polypeptide sequence are madethrough mutagenesis of the underlying polynucleotides that encode them.These techniques provides a straightforward approach to prepare and testsequence variants, for example, incorporating one or more of theforegoing considerations, by introducing one or more nucleotide sequencechanges into the polynucleotide. Site-specific mutagenesis allows theproduction of mutants through the use of specific oligonucleotidesequences include the nucleotide sequence of the desired mutation, aswell as a sufficient number of adjacent nucleotides, to provide a primersequence of sufficient size and sequence complexity to form a stableduplex on both sides of the deletion junction being traversed. Mutationsare employed in a selected polynucleotide sequence to improve, alter,decrease, modify, or otherwise change the properties of thepolynucleotide itself, and/or alter the properties, activity,composition, stability, or primary sequence of the encoded polypeptide.

In other embodiments of the present invention, the polynucleotidesequences provided herein are used as probes or primers for nucleic acidhybridization, e.g., as PCR primers. The ability of such nucleic acidprobes to specifically hybridize to a sequence of interest enable themto detect the presence of complementary sequences in a given sample.However, other uses are also encompassed by the invention, such as theuse of the sequence information for the preparation of mutant speciesprimers, or primers for use in preparing other genetic constructions. Assuch, nucleic acid segments of the invention that include a sequenceregion of at least about 15 nucleotide long contiguous sequence that hasthe same sequence as, or is complementary to, a 15 nucleotide longcontiguous sequence disclosed herein is particularly useful. Longercontiguous identical or complementary sequences, e.g., those of about20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths)including full length sequences, and all lengths in between, are alsoused in certain embodiments.

Polynucleotide molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so (including intermediate lengths as well),identical or complementary to a polynucleotide sequence disclosedherein, are particularly contemplated as hybridization probes for usein, e.g., Southern and Northern blotting, and/or primers for use in,e.g., polymerase chain reaction (PCR). The total size of fragment, aswell as the size of the complementary stretch(es), ultimately depends onthe intended use or application of the particular nucleic acid segment.Smaller fragments are generally used in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 15 and about 100 nucleotides, but largercontiguous complementarity stretches may be used, according to thelength complementary sequences one wishes to detect.

The use of a hybridization probe of about 15-25 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 12 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. Nucleic acid molecules having gene-complementary stretches of15 to 25 contiguous nucleotides, or even longer where desired, aregenerally preferred.

Hybridization probes are selected from any portion of any of thesequences disclosed herein. All that is required is to review thesequences set forth herein, or to any continuous portion of thesequences, from about 15-25 nucleotides in length up to and includingthe full length sequence, that one wishes to utilize as a probe orprimer. The choice of probe and primer sequences is governed by variousfactors. For example, one may wish to employ primers from towards thetermini of the total sequence.

Polynucleotide of the present invention, or fragments or variantsthereof, are readily prepared by, for example, directly synthesizing thefragment by chemical means, as is commonly practiced using an automatedoligonucleotide synthesizer. Also, fragments are obtained by applicationof nucleic acid reproduction technology, such as the PCR™ technology ofU.S. Pat. No. 4,683,202, by introducing selected sequences intorecombinant vectors for recombinant production, and by other recombinantDNA techniques generally known to those of skill in the art of molecularbiology.

Vectors, Host Cells and Recombinant Methods

The invention provides vectors and host cells comprising a nucleic acidof the present invention, as well as recombinant techniques for theproduction of a polypeptide of the present invention. Vectors of theinvention include those capable of replication in any type of cell ororganism, including, e.g., plasmids, phage, cosmids, and minichromosomes. In various embodiments, vectors comprising a polynucleotideof the present invention are vectors suitable for propagation orreplication of the polynucleotide, or vectors suitable for expressing apolypeptide of the present invention. Such vectors are known in the artand commercially available.

Polynucleotides of the present invention are synthesized, whole or inparts that are then combined, and inserted into a vector using routinemolecular and cell biology techniques, including, e.g., subcloning thepolynucleotide into a linearized vector using appropriate restrictionsites and restriction enzymes. Polynucleotides of the present inventionare amplified by polymerase chain reaction using oligonucleotide primerscomplementary to each strand of the polynucleotide. These primers alsoinclude restriction enzyme cleavage sites to facilitate subcloning intoa vector. The replicable vector components generally include, but arenot limited to, one or more of the following: a signal sequence, anorigin of replication, and one or more marker or selectable genes.

In order to express a polypeptide of the present invention, thenucleotide sequences encoding the polypeptide, or functionalequivalents, are inserted into an appropriate expression vector, i.e., avector that contains the necessary elements for the transcription andtranslation of the inserted coding sequence. Methods well known to thoseskilled in the art are used to construct expression vectors containingsequences encoding a polypeptide of interest and appropriatetranscriptional and translational control elements. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination. Such techniques are described, forexample, in Sambrook, J., et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York. N.Y.

A variety of expression vector/host systems are utilized to contain andexpress polynucleotide sequences. These include, but are not limited to,microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Withinone embodiment, the variable regions of a gene expressing a monoclonalantibody of interest are amplified from a hybridoma cell usingnucleotide primers. These primers aer synthesized by one of ordinaryskill in the art, or may be purchased from commercially availablesources (see, e.g., Stratagene (La Jolla, Calif.), which sells primersfor amplifying mouse and human variable regions. The primers are used toamplify heavy or light chain variable regions, which are then insertedinto vectors such as ImmunoZAP™ H or ImmunoZAP™ L (Stratagene),respectively. These vectors are then introduced into E. coli, yeast, ormammalian-based systems for expression. Large amounts of a single-chainprotein containing a fusion of the V_(H) and V_(L) domains are producedusing these methods (see Bird et al., Science 242:423-426 (1988)).

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of the vector, e.g.,enhancers, promoters, 5′ and 3′ untranslated regions, that interact withhost cellular proteins to carry out transcription and translation. Suchelements may vary in their strength and specificity. Depending on thevector system and host utilized, any number of suitable transcriptionand translation elements, including constitutive and induciblepromoters, are used.

Examples of promoters suitable for use with prokaryotic hosts includethe phoa promoter, β-lactamase and lactose promoter systems, alkalinephosphatase promoter, a tryptophan (trp) promoter system, and hybridpromoters such as the tac promoter. However, other known bacterialpromoters are suitable. Promoters for use in bacterial systems alsousually contain a Shine-Dalgarno sequence operably linked to the DNAencoding the polypeptide. Inducible promoters such as the hybrid lacZpromoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) orPSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) and the like are used.

A variety of promoter sequences are known for eukaryotes and any areused according to the present invention. Virtually all eukaryotic geneshave an AT-rich region located approximately 25 to 30 bases upstreamfrom the site where transcription is initiated. Another sequence found70 to 80 bases upstream from the start of transcription of many genes isa CNCAAT region where N may be any nucleotide. At the 3′ end of mosteukaryotic genes is an AATAAA sequence that may be the signal foraddition of the poly A tail to the 3′ end of the coding sequence. All ofthese sequences are suitably inserted into eukaryotic expressionvectors.

In mammalian cell systems, promoters from mammalian genes or frommammalian viruses are generally preferred. Polypeptide expression fromvectors in mammalian host cells aer controlled, for example, bypromoters obtained from the genomes of viruses such as polyoma virus,fowlpox virus, adenovirus (e.g., Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-Bvirus and most preferably Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, and from heat-shock promoters, provided such promoters arecompatible with the host cell systems. If it is necessary to generate acell line that contains multiple copies of the sequence encoding apolypeptide, vectors based on SV40 or EBV may be advantageously usedwith an appropriate selectable marker. One example of a suitableexpression vector is pcDNA-3.1 (Invitrogen, Carlsbad, Calif.), whichincludes a CMV promoter.

A number of viral-based expression systems are available for mammalianexpression of polypeptides. For example, in cases where an adenovirus isused as an expression vector, sequences encoding a polypeptide ofinterest may be ligated into an adenovirus transcription/translationcomplex consisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus that is capable of expressing thepolypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc.Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

In bacterial systems, any of a number of expression vectors are selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities are desired, vectors that direct highlevel expression of fusion proteins that are readily purified are used.Such vectors include, but are not limited to, the multifunctional E.coli cloning and expression vectors such as BLUESCRIPT (Stratagene), inwhich the sequence encoding the polypeptide of interest may be ligatedinto the vector in frame with sequences for the amino-terminal Met andthe subsequent 7 residues of β-galactosidase, so that a hybrid proteinis produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega,Madison, Wis.) are also used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems are designedto include heparin, thrombin, or factor XA protease cleavage sites sothat the cloned polypeptide of interest can be released from the GSTmoiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH are used. Examples of other suitable promoter sequencesfor use with yeast hosts include the promoters for 3-phosphoglyceratekinase or other glycolytic enzymes, such as enolase,glyceraldehyde-3-phosphate dehydrogcnase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. For reviews, see Ausubel etal. (supra) and Grant et al. (1987) Methods Enzymol. 153:516-544. Otheryeast promoters that are inducible promoters having the additionaladvantage of transcription controlled by growth conditions include thepromoter regions for alcohol dehydrogenase 2, isocytochrome C, acidphosphatase, degradative enzymes associated with nitrogen metabolism,metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Suitable vectors andpromoters for use in yeast expression are further described in EP73,657. Yeast enhancers also are advantageously used with yeastpromoters.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides are driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV are used alone or in combination with the omega leadersequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters are used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J., et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, e.g., Hobbs,S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology(1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system is also used to express a polypeptide of interest. Forexample, in one such system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding thepolypeptide are cloned into a non-essential region of the virus, such asthe polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of the polypeptide-encoding sequencerenders the polyhedrin gene inactive and produce recombinant viruslacking coat protein. The recombinant viruses are then used to infect,for example, S. frugiperda cells or Trichoplusia larvae, in which thepolypeptide of interest is expressed (Engelhard, E. K. et al. (1994)Proc. Natl. Acad. Sci. 91:3224-3227).

Specific initiation signals are also used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon are provided. Furthermore, theinitiation codon is in the correct reading frame to ensure correcttranslation of the inserted polynucleotide. Exogenous translationalelements and initiation codons are of various origins, both natural andsynthetic.

Transcription of a DNA encoding a polypeptide of the invention is oftenincreased by inserting an enhancer sequence into the vector. Manyenhancer sequences are known, including, e.g., those identified in genesencoding globin, elastase, albumin, α-fetoprotein, and insulin.Typically, however, an enhancer from a eukaryotic cell virus is used.Examples include the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) onenhancing elements for activation of eukaryotic promoters. The enhanceris spliced into the vector at a position 5′ or 3′ to thepolypeptide-encoding sequence, but is preferably located at a site 5′from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) typically also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding anti-PSCA antibody. Oneuseful transcription termination component is the bovine growth hormonepolyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, plant or higher eukaryote cellsdescribed above. Examples of suitable prokaryotes for this purposeinclude eubacteria, such as Gram-negative or Gram-positive organisms,for example, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as wellas Bacilli such as B. subtilis and B. licheniformis (e.g., B.licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989),Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E.coli cloning host is E. coli 294 (ATCC 31,446), although other strainssuch as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC27,325) are suitable. These examples are illustrative rather thanlimiting.

Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among lower eukaryotic host microorganisms. However, a number ofother genera, species, and strains are commonly available and usedherein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as,e.g., K lactis, K fragilis (ATCC 12,424), K bulgaricus (ATCC 16,045), Kwickeramii (ATCC 24,178), K waltii (ATCC 56,500), K drosophilarum (ATCC36,906), K thermotolerans, and K marxianus; yarrowia (EP 402,226);Pichia pastoris. (EP 183,070); Candida; Trichoderma reesia (EP 244,234);Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis;and filamentous fungi such as, e.g., Neurospora, Penicillium,Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

In certain embodiments, a host cell strain is chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation.glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing that cleaves a “prepro” form of theprotein is also used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, COS, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, are chosen to ensurethe correct modification and processing of the foreign protein.

Methods and reagents specifically adapted for the expression ofantibodies or fragments thereof are also known and available in the art,including those described, e.g., in U.S. Pat. Nos. 4,816,567 and6,331,415. In various embodiments, antibody heavy and light chains, orfragments thereof, are expressed from the same or separate expressionvectors. In one embodiment, both chains are expressed in the same cell,thereby facilitating the formation of a functional antibody or fragmentthereof.

Full length antibody, antibody fragments, and antibody fusion proteinsare produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) and theimmunoconjugate by itself shows effectiveness in infected celldestruction. For expression of antibody fragments and polypeptides inbacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523,which describes translation initiation region (TIR) and signal sequencesfor optimizing expression and secretion. After expression, the antibodyis isolated from the E. coli cell paste in a soluble fraction and can bepurified through, e.g., a protein A or G column depending on theisotype. Final purification can be carried out using a process similarto that used for purifying antibody expressed e.g., in CHO cells.

Suitable host cells for the expression of glycosylated polypeptides andantibodies are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopicius (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses are used as the virus herein according to the presentinvention, particularly for transfection of Spodoptera frugiperda cells.Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco are also utilized as hosts.

Methods of propagation of antibody polypeptides and fragments thereof invertebrate cells in culture (tissue culture) are encompassed by theinvention. Examples of mammalian host cell lines used in the methods ofthe invention are monkey kidney CV1 line transformed by SV40 (COS-7,ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subclonedfor growth in suspension culture, Graham et al., J. Gen Virol. 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for polypeptide production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines that stablyexpress a polynucleotide of interest are transformed using expressionvectors that contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells areallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells that successfully express the introduced sequences.Resistant clones of stably transformed cells are proliferated usingtissue culture techniques appropriate to the cell type.

A plurality of selection systems are used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) genesthat are employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance is used as the basisfor selection; for example, dhfr, which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin, F. et al. (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described. For example, trpBallows cells to utilize indole in place of tryptophan, and hisD allowscells to utilize histinol in place of histidine (Hartman, S. C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51). The use ofvisible markers has gained popularity with such markers as anthocyanins,beta-glucuronidase and its substrate GUS, and luciferase and itssubstrate luciferin, being widely used not only to identifytransformants, but also to quantify the amount of transient or stableprotein expression attributable to a specific vector system (Rhodes, C.A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression isconfirmed. For example, if the sequence encoding a polypeptide isinserted within a marker gene sequence, recombinant cells containingsequences are identified by the absence of marker gene function.Alternatively, a marker gene is placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well. Alternatively,host cells that contain and express a desired polynucleotide sequenceare identified by a variety of procedures known to those of skill in theart. These procedures include, but are not limited to, DNA-DNA orDNA-RNA hybridizations and protein bioassay or immunoassay techniqueswhich include, for example, membrane, solution, or chip basedtechnologies for the detection and/or quantification of nucleic acid orprotein.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Nonlimitingexamples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).A two-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering epitopes on a given polypeptide ispreferred for some applications, but a competitive binding assay mayalso be employed. These and other assays are described, among otherplaces, in Hampton, R. et al. (1990; Serological Methods, a LaboratoryManual, APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983; J.Exp. Med. 158:1211-1216).

Various labels and conjugation techniques are known by those skilled inthe art and are used in various nucleic acid and amino acid assays.Means for producing labeled hybridization or PCR probes for detectingsequences related to polynucleotides include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof arecloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and are used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures are conducted using a variety of commercially available kits.Suitable reporter molecules or labels, which are used include, but arenot limited to, radionuclides, enzymes, fluorescent, chemiluminescent,or chromogenic agents as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

The polypeptide produced by a recombinant cell is secreted or containedintracellularly depending on the sequence and/or the vector used.Expression vectors containing polynucleotides of the invention aredesigned to contain signal sequences that direct secretion of theencoded polypeptide through a prokaryotic or eukaryotic cell membrane.

In certain embodiments, a polypeptide of the invention is produced as afusion polypeptide further including a polypeptide domain thatfacilitates purification of soluble proteins. Suchpurification-facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Amgen, Seattle,Wash.). The inclusion of cleavable linker sequences such as thosespecific for Factor XA or enterokinase (Invitrogen. San Diego, Calif.)between the purification domain and the encoded polypeptide are used tofacilitate purification. An exemplary expression vector provides forexpression of a fusion protein containing a polypeptide of interest anda nucleic acid encoding 6 histidine residues preceding a thioredoxin oran enterokinase cleavage site. The histidine residues facilitatepurification on IMIAC (immobilized metal ion affinity chromatography) asdescribed in Porath, J. et al. (1992, Prot. Exp. Purif 3:263-281) whilethe enterokinase cleavage site provides a means for purifying thedesired polypeptide from the fusion protein. A discussion of vectorsused for producing fusion proteins is provided in Kroll, D. J. et al.(1993; DNA Cell Biol. 12:441-453).

In certain embodiments, a polypeptide of the present invention is fusedwith a heterologous polypeptide, which may be a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature protein or polypeptide. The heterologous signal sequence selectedpreferably is one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For prokaryotic host cells, thesignal sequence is selected, for example, from the group of the alkalinephosphatase, penicillinase, 1pp, or heat-stable enterotoxin II leaders.For yeast secretion, the signal sequence is selected from, e.g., theyeast invertase leader, α factor leader (including Saccharomyces andKluyveromyces α factor leaders), or acid phosphatase leader, the C.albicans glucoamylase leader, or the signal described in WO 90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable.

When using recombinant techniques, the polypeptide or antibody isproduced intracellularly, in the periplasmic space, or directly secretedinto the medium. If the polypeptide or antibody is producedintracellularly, as a first step, the particulate debris, either hostcells or lysed fragments, are removed, for example, by centrifugation orultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992)describe a procedure for isolating antibodies that are secreted to theperiplasmic space of E. coli. Briefly, cell paste is thawed in thepresence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris isremoved by centrifugation. Where the polypeptide or antibody is secretedinto the medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Optionally, a protease inhibitor such as PMSF is included in anyof the foregoing steps to inhibit proteolysis and antibiotics isincluded to prevent the growth of adventitious contaminants.

The polypeptide or antibody composition prepared from the cells arepurified using, for example, hydroxylapatite chromatography, gelelectrophoresis, dialysis, and affinity chromatography, with affinitychromatography being the preferred purification technique. Thesuitability of protein A as an affinity ligand depends on the speciesand isotype of any immunoglobulin Fc domain that is present in thepolypeptide or antibody. Protein A is used to purify antibodies orfragments thereof that are based on human γ₁, γ₂, or γ₄ heavy chains(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ₃ (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where thepolypeptide or antibody comprises a C_(H)3 domain, the Bakerbond ABX™resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.Other techniques for protein purification such as fractionation on anion-exchange column, ethanol precipitation, Reverse Phase HPLC,chromatography on silica, chromatography on heparin SEPHAROSE™chromatography on an anion or cation exchange resin (such as apolyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the polypeptide orantibody to be recovered. Following any preliminary purificationstep(s), the mixture comprising the polypeptide or antibody of interestand contaminants are subjected to low pH hydrophobic interactionchromatography using an elution buffer at a pH between about 2.5-4.5,preferably performed at low salt concentrations (e.g., from about0-0.25M salt).

Pharmaceutical Compositions

The invention further includes pharmaceutical formulations including apolypeptide, antibody, or modulator of the present invention, at adesired degree of purity, and a pharmaceutically acceptable carrier,excipient, or stabilizer (Remingion's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980)). In certain embodiments, pharmaceuticalformulations are prepared to enhance the stability of the polypeptide orantibody during storage, e.g., in the form of lyophilized formulationsor aqueous solutions. Acceptable carriers, excipients, or stabilizersare nontoxic to recipients at the dosages and concentrations employed,and include, e.g., buffers such as acetate, Tris, phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;tonicifiers such as trehalose and sodium chloride; sugars such assucrose, mannitol, trehalose or sorbitol; surfactant such aspolysorbate; salt-forming counter-ions such as sodium; metal complexes(e.g. Zn-protein complexes); and/or non-ionic surfactants such asTWEEN™, PLURONICS™ or polyethylene glycol (PEG). In certain embodiments,the therapeutic formulation preferably comprises the polypeptide orantibody at a concentration of between 5-200 mg/ml, preferably between10-100 mg/ml.

The formulations herein also contain one or more additional therapeuticagents suitable for the treatment of the particular indication, e.g.,infection being treated, or to prevent undesired side-effects.Preferably, the additional therapeutic agent has an activitycomplementary to the polypeptide or antibody of the resent invention,and the two do not adversely affect each other. For example, in additionto the polypeptide or antibody of the invention, an additional or secondantibody, anti-viral agent, anti-infective agent and/or cardioprotectantis added to the formulation. Such molecules are suitably present in thepharmaceutical formulation in amounts that are effective for the purposeintended.

The active ingredients, e.g., polypeptides and antibodies of theinvention and other therapeutic agents, are also entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and polymethylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemingion's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations are prepared. Suitable examples ofsustained-release preparations include, but are not limited to,semi-permeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Nonlimiting examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxyburyric acid.

Formulations to be used for in vivo administration are preferablysterile. This is readily accomplished by filtration through sterilefiltration membranes.

Diagnostic Uses

Antibodies and fragments thereof, and therapeutic compositions, of theinvention specifically bind or preferentially bind to infected cells ortissue, as compared to normal control cells and tissue. Thus, theseinfluenza A antibodies are used to detect infected cells or tissues in apatient, biological sample, or cell population, using any of a varietyof diagnostic and prognostic methods, including those described herein.The ability of an anti-M2e specific antibody to detect infected cellsdepends upon its binding specificity, which is readily determined bytesting its ability to bind to infected cells or tissues obtained fromdifferent patients, and/or from patients infected with different strainsof Influenza A. Diagnostic methods generally involve contacting abiological sample obtained from a patient, such as, e.g., blood, serum,saliva, urine, sputum, a cell swab sample, or a tissue biopsy, with anInfluenza A, e.g., HuM2e antibody and determining whether the antibodypreferentially binds to the sample as compared to a control sample orpredetermined cut-off value, thereby indicating the presence of infectedcells. In particular embodiments, at least two-fold, three-fold, orfive-fold more HuM2e antibody binds to an infected cell as compared toan appropriate control normal cell or tissue sample. A pre-determinedcut-off value is determined, e.g., by averaging the amount of HuM2eantibody that binds to several different appropriate control samplesunder the same conditions used to perform the diagnostic assay of thebiological sample being tested.

Bound antibody is detected using procedures described herein and knownin the art. In certain embodiments, diagnostic methods of the inventionare practiced using HuM2e antibodies that are conjugated to a detectablelabel, e.g., a fluorophore, to facilitate detection of bound antibody.However, they are also practiced using methods of secondary detection ofthe HuM2e antibody. These include, for example, RIA, ELISA,precipitation, agglutination, complement fixation andimmuno-fluorescence.

In certain procedures, the HuM2e antibodies are labeled. The label isdetected directly. Exemplary labels that are detected directly include,but are not limited to, radiolabels and fluorochromes. Alternatively, orin addition, labels are moieties, such as enzymes, that must be reactedor derivatized to be detected. Nonlimiting examples of isotope labelsare ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P and ³⁵S. Fluorescent materials thatare used include, but are not limited to, for example, fluorescein andits derivatives, rhodamine and its derivatives, auramine, dansyl,umbelliferone, luciferia, 2,3-dihydrophthalazinediones, horseradishperoxidase, alkaline phosphatase, lysozyme, and glucose-6-phosphatedehydrogenase.

An enzyme label is detected by any of the currently utilizedcolorimetric, spectrophotometric, fluorospectro-photometric orgasometric techniques. Many enzymes which are used in these proceduresare known and utilized by the methods of the invention. Nonlimitingexamples are peroxidase, alkaline phosphatase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase, galactose oxidase plus peroxidase and acid phosphatase.

The antibodies are tagged with such labels by known methods. Forinstance, coupling agents such as aldehydes, carbodiimides, dimaleimide,imidates, succinimides, bid-diazotized benzadine and the like are usedto tag the antibodies with the above-described fluorescent,chemiluminescent, and enzyme labels. An enzyme is typically combinedwith an antibody using bridging molecules such as carbodiimides,periodate, diisocyanates, glutaraldehyde and the like. Various labelingtechniques are described in Morrison, Methods in Enzymology 32b, 103(1974), Syvanen et al., J. Biol. Chem. 284, 3762 (1973) and Bolton andHunter, Biochem J. 133, 529 (1973).

HuM2e antibodies of the present invention are capable of differentiatingbetween patients with and patients without an Influenza A infection, anddetermining whether or not a patient has an infection, using therepresentative assays provided herein. According to one method, abiological sample is obtained from a patient suspected of having orknown to have an Influenza A infection. In preferred embodiments, thebiological sample includes cells from the patient. The sample iscontacted with an HuM2e antibody, e.g., for a time and under conditionssufficient to allow the HuM2e antibody to bind to infected cells presentin the sample. For instance, the sample is contacted with an HuM2eantibody for 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 30minutes, 1 hour, 6 hours, 12 hours, 24 hours, 3 days or any point inbetween. The amount of bound HuM2e antibody is determined and comparedto a control value, which may be, e.g., a predetermined value or a valuedetermined from normal tissue sample. An increased amount of antibodybound to the patient sample as compared to the control sample isindicative of the presence of infected cells in the patient sample.

In a related method, a biological sample obtained from a patient iscontacted with an HuM2e antibody for a time and under conditionssufficient to allow the antibody to bind to infected cells. Boundantibody is then detected, and the presence of bound antibody indicatesthat the sample contains infected cells. This embodiment is particularlyuseful when the HuM2e antibody does not bind normal cells at adetectable level. Different HuM2e antibodies possess different bindingand specificity characteristics. Depending upon these characteristics,particular HuM2e antibodies are used to detect the presence of one ormore strains of Influenza A. For example, certain antibodies bindspecifically to only one or several strains of Influenza virus, whereasothers bind to all or a majority of different strains of Influenzavirus. Antibodies specific for only one strain of Influenza A are usedto identify the strain of an infection.

In certain embodiments, antibodies that bind to an infected cellpreferably generate a signal indicating the presence of an infection inat least about 20% of patients with the infection being detected, morepreferably at least about 30% of patients. Alternatively, or inaddition, the antibody generates a negative signal indicating theabsence of the infection in at least about 90% of individuals withoutthe infection being detected. Each antibody satisfies the abovecriteria; however, antibodies of the present invention are used incombination to improve sensitivity.

The present invention also includes kits useful in performing diagnosticand prognostic assays using the antibodies of the present invention.Kits of the invention include a suitable container comprising a HuM2eantibody of the invention in either labeled or unlabeled form. Inaddition, when the antibody is supplied in a labeled form suitable foran indirect binding assay, the kit further includes reagents forperforming the appropriate indirect assay. For example, the kit includesone or more suitable containers including enzyme substrates orderivatizing agents, depending on the nature of the label. Controlsamples and/or instructions are also included.

Therapeutic/Prophylactic Uses

Passive immunization has proven to be an effective and safe strategy forthe prevention and treatment of viral diseases. (See Keller et al.,Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall, Nat. Biotechnol.20:114 (2002); Shibata et al., Nat. Med. 5:204-10 (1999); and Igarashiet al., Nat. Med. 5:211-16 (1999), each of which are incorporated hereinby reference)). Passive immunization using human monoclonal antibodiesprovide an immediate treatment strategy for emergency prophylaxis andtreatment of influenza

HuM2e antibodies and fragments thereof, and therapeutic compositions, ofthe invention specifically bind or preferentially bind to infectedcells, as compared to normal control uninfected cells and tissue. Thus,these HuM2e antibodies are used to selectively target infected cells ortissues in a patient, biological sample, or cell population. In light ofthe infection-specific binding properties of these antibodies, thepresent invention provides methods of regulating (e.g., inhibiting) thegrowth of infected cells, methods of killing infected cells, and methodsof inducing apoptosis of infected cells. These methods includecontacting an infected cell with an HuM2e antibody of the invention.These methods are practiced in vitro, ex vivo, and in vivo.

In various embodiments, antibodies of the invention are intrinsicallytherapeutically active. Alternatively, or in addition, antibodies of theinvention are conjugated to a cytotoxic agent or growth inhibitoryagent, e.g., a radioisotope or toxin, that is used in treating infectedcells bound or contacted by the antibody.

In one embodiment, the invention provides methods of treating orpreventing infection in a patient, including the steps of providing anHuM2e antibody of the invention to a patient diagnosed with, at risk ofdeveloping, or suspected of having an Influenza A infection. The methodsof the invention are used in the first-line treatment of the infection,follow-on treatment, or in the treatment of a relapsed or refractoryinfection. Treatment with an antibody of the invention is a stand alonetreatment. Alternatively, treatment with an antibody of the invention isone component or phase of a combination therapy regime, in which one ormore additional therapeutic agents are also used to treat the patient.

Subjects at risk for an influenza virus-related diseases or disordersinclude patients who have come into contact with an infected person orwho have been exposed to the influenza virus in some other way.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the influenza virus-relateddisease or disorder, such that a disease or disorder is prevented or,alternatively, delayed in its progression.

In various aspects, the huM2e is administered substantiallycontemporaneously with or following infection of the subject, i.e.,therapeutic treatment. In another aspect, the antibody provides atherapeutic benefit. In various aspects, a therapeutic benefit includesreducing or decreasing progression, severity, frequency, duration orprobability of one or more symptoms or complications of influenzainfection, virus titer, virus replication or an amount of a viralprotein of one or more influenza strains. still another aspect, atherapeutic benefit includes hastening or accelerating a subject'srecovery from influenza infection.

Methods for preventing an increase in influenza virus titer, virusreplication, virus proliferation or an amount of an influenza viralprotein in a subject are further provided. In one embodiment, a methodincludes administering to the subject an amount of a huM2e antibodyeffective to prevent an increase in influenza virus titer, virusreplication or an amount of an influenza viral protein of one or moreinfluenza strains or isolates in the subject.

Methods for protecting a subject from infection or decreasingsusceptibility of a subject to infection by one or more influenzastrains/isolates or subtypes, i.e., prophylactic methods, areadditionally provided. In one embodiment, a method includesadministering to the subject an amount of huM2e antibody thatspecifically binds influenza M2 effective to protect the subject frominfection, or effective to decrease susceptibility of the subject toinfection, by one or more influenza strains/isolates or subtypes.

Optionally, the subject is further administered with a second agent suchas, but not limited to, an influenza virus antibody, an anti-viral drugsuch as a neuraminidase inhibitor, a HA inhibitor, a sialic acidinhibitor or an M2 ion channel inhibitor, a viral entry inhibitor or aviral attachment inhibitor. The M2 ion channel inhibitor is for exampleamantadine or rimantadine. The neuraminidase inhibitor for examplezanamivir, or oseltamivir phosphate.

Symptoms or complications of influenza infection that can be reduced ordecreased include, for example, chills, fever, cough, sore throat, nasalcongestion, sinus congestion, nasal infection, sinus infection, bodyache, head ache, fatigue, pneumonia, bronchitis, ear infection, ear acheor death.

For in vivo treatment of human and non-human patients, the patient isusually administered or provided a pharmaceutical formulation includinga HuM2e antibody of the invention. When used for in vivo therapy, theantibodies of the invention are administered to the patient intherapeutically effective amounts (i.e., amounts that eliminate orreduce the patient's viral burden). The antibodies are administered to ahuman patient, in accord with known methods, such as intravenousadministration, e.g., as a bolus or by continuous infusion over a periodof time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. The antibodies may be administeredparenterally, when possible, at the target cell site, or intravenously.Intravenous or subcutaneous administration of the antibody is preferredin certain embodiments. Therapeutic compositions of the invention areadministered to a patient or subject systemically, parenterally, orlocally.

For parenteral administration, the antibodies are formulated in a unitdosage injectable form (solution, suspension, emulsion) in associationwith a pharmaceutically acceptable, parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Nonaqueous vehicles such as fixed oils and ethyloleate are also used. Liposomes are used as carriers. The vehiclecontains minor amounts of additives such as substances that enhanceisotonicity and chemical stability, e.g., buffers and preservatives. Theantibodies are typically formulated in such vehicles at concentrationsof about 1 mg/ml to 10 mg/ml.

The dose and dosage regimen depends upon a variety of factors readilydetermined by a physician, such as the nature of the infection and thecharacteristics of the particular cytotoxic agent or growth inhibitoryagent conjugated to the antibody (when used), e.g., its therapeuticindex, the patient, and the patient's history. Generally, atherapeutically effective amount of an antibody is administered to apatient. In particular embodiments, the amount of antibody administeredis in the range of about 0.1 mg/kg to about 50 mg/kg of patient bodyweight. Depending on the type and severity of the infection, about 0.1mg/kg to about 50 mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) ofantibody is an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. The progress of this therapy is readilymonitored by conventional methods and assays and based on criteria knownto the physician or other persons of skill in the art.

In one particular embodiment, an immunoconjugate including the antibodyconjugated with a cytotoxic agent is administered to the patient.Preferably, the immunoconjugate is internalized by the cell, resultingin increased therapeutic efficacy of the immunoconjugate in killing thecell to which it binds. In one embodiment, the cytotoxic agent targetsor interferes with the nucleic acid in the infected cell. Examples ofsuch cytotoxic agents are described above and include, but are notlimited to, maytansinoids, calicheamicins, ribonucleases and DNAendonucleases.

Other therapeutic regimens are combined with the administration of theHuM2e antibody of the present invention. The combined administrationincludes co-administration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder, wherein preferably there is a time period while both (or all)active agents simultaneously exert their biological activities.Preferably such combined therapy results in a synergistic therapeuticeffect.

In certain embodiments, it is desirable to combine administration of anantibody of the invention with another antibody directed against anotherantigen associated with the infectious agent.

Aside from administration of the antibody protein to the patient, theinvention provides methods of administration of the antibody by genetherapy. Such administration of nucleic acid encoding the antibody isencompassed by the expression “administering a therapeutically effectiveamount of an antibody”. See, for example, PCT Patent ApplicationPublication WO96/07321 concerning the use of gene therapy to generateintracellular antibodies.

In another embodiment, anti-M2e antibodies of the invention are used todetermine the structure of bound antigen, e.g., conformational epitopes,the structure of which is then used to develop a vaccine having ormimicking this structure, e.g., through chemical modeling and SARmethods. Such a vaccine could then be used to prevent Influenza Ainfection.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheetare incorporated herein byreference, in their entirety.

EXAMPLES Example 1 Screening and Characterization of M2e-specificAntibodies Present in Human Plasma Using Cells Expressing RecombinantM2e Protein

Fully human monoclonal antibodies specific for M2 and capable of bindingto influenza A infected cells and the influenza virus itself wereidentified in patient serum, as described below.

Expression of M2 in Cell Lines

An expression construct containing the M2 full length cDNA,corresponding to the derived M2 sequence found in Influenza subtypeH₃N₂, was transfected into 293 cells.

The M2 cDNA is encoded by the following polynucleotide sequence and SEQID NO: 53:

ATGAGTCTTCTAACCGAGGTCGAAACGCCTATCAGAAACGAATGGGGGTGCAGATGCAACGATTCAAGTGATCCTCTTGTTGTTGCCGCAAGTATCATTGGGATCCTGCACTTGATATTGTGGATTCTTGATCGTCTTTTTTTCAAATGCATTTATCGTCTCTTTAAACACGGTCTGAAAAGAGGGCCTTCTACGGAAGGAGTACCAGAGTCTATGAGGGAAGAATATCGAAAGGAACAGCAGAGTGCTGTGGATGCTGACGATAGTCATTTTGTCAACATAGAGCTGGAG

The cell surface expression of M2 was confirmed using the anti-M2epeptide specific MAb 14C2. Two other variants of M2, from A/HongKong/483/1997 (HK483) and A/Vietnam/1203/2004 (VN1203), were used forsubsequent analyses, and their expression was determined usingM2e-specific monoclonal antibodies of the present invention, since 14C2binding may be abrogated by the various amino acid substitutions in M2e.

Screening of Antibodies in Peripheral Blood

Over 120 individual plasma samples were tested for antibodies that boundM2. None of them exhibited specific binding to the M2e peptide. However,10% of the plasma samples contained antibodies that bound specificallyto the 293-M2H3N₂ cell line. This indicates that the antibodies could becategorized as binding to conformational determinants of an M2homotetramer, and binding to conformational determinants of multiplevariants of the M2 homotetramer; they could not be specific for thelinear M2e peptide.

Characterization of Anti-M2 MAbs

The human MAbs identified through this process proved to bind toconformational epitopes on the M2 homotetramer. They bound to theoriginal 293-M2 transfectant, as well as to the two other cell-expressedM2 variants. The 14C2 MAb, in addition to binding the M2e peptide,proved to be more sensitive to the M2 variant sequences. Moreover, 14C2does not readily bind influenza virions, while the conformation specificanti-M2 MAbs did.

These results demonstrate that the methods of the invention provide forthe identification of M2 MAbs from normal human immune responses toinfluenza without a need for specific immunization of M2. If used forimmunotherapy, these fully human MAbs have the potential to be bettertolerated by patients that humanized mouse antibodies. Additionally, andin contrast to 14C2 and the Gemini Biosciences MAbs, which bind tolinear M2e peptide, the MAbs of the invention bind to conformationalepitopes of M2, and are specific not only for cells infected with Astrain influenza, but also for the virus itself. Another advantage ofthe MAbs of the invention is that they each bind all of the M2 variantsyet tested, indicating that they are not restricted to a specific linearamino acid sequence.

Example 2 Identification of M2-Specific Antibodies

Mononuclear or B cells expressing three of the MAbs identified in humanserum as described in Example 1 were diluted into clonal populations andinduced to produce antibodies. Antibody containing supernatants werescreened for binding to 293 FT cells stably transfected with the fulllength M2E protein from influenza strain Influenza subtype H3N2.Supernatants which showed positive staining/binding were re-screenedagain on 293 FT cells stably transfected with the full length M2Eprotein from influenza strain Influenza subtype H3N₂ and on vector alonetransfected cells as a control.

The variable regions of the antibodies were then rescue cloned from theB cell wells whose supernatants showed positive binding. Transienttransfections were performed in 293 FT cells to reconstitute and producethese antibodies. Reconstituted antibody supernatants were screened forbinding to 293 FT cells stably transfected with the full length M2Eprotein as detailed above to identify the rescued anti-M2E antibodies.Three different antibodies were identified: 8i10, 21B15 and 23K12. Afourth additional antibody clone was isolated by the rescue screens,4C2. However, it was not unique and had the exact same sequence as clone8i10 even though it came from a different donor than clone 8i10.

The sequences of the kappa and gamma variable regions of theseantibodies are provided below.

Clone 8i10:

The Kappa LC variable region of the anti M2 clone 8i10 was cloned asHind III to BsiW1 fragment (see below), and is encoded by the followingpolynucleotide sequences, and SEQ ID NO: 54 (top) and SEQ ID NO: 55(bottom):

HindIIIAAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGTTCGAAGGTGGTACCTGTACTCCCAGGAGCGAGTCGAGGACCCCGAGGACGATGAGACCGAGGCTCCACCCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAGGTCTACACTGTAGGTCTACTGGGTCAGAGGTAGGAGGGACAGACGTAGACATCCTCTGTCTCAGTGGTTCACTTGCCGGGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCCCAGTGAACGGCCCGCTCAGTCTTGTAAATGTTCATAAATTTAACCATAGTCGTCTCTGGTCCCTTTCGGGCTAAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATGATTCCCGGACTAGAGACGACGTAGGCCCAACGTTTCACCCCAGGGTAGTTCCAAGTCACCGTCACCTACTGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACGACCCTGTCTAAAGTGAGAGTGGTAGTGGTCAGACGTTGGACTTCTAAAACGTTGAATGATGACAGTTG                                                     BsiWIAGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGGTGGAGATCAAACGTACGTCTCAATGTCAGGGGGAGAGTGAAAGCCGCCTCCCTGGTCCCACCTCTAGTTTGCATGC

The translation of the 8i10 Kappa LC variable region is as follows,polynucleotide sequence (above, SEQ ID NO: 54, top) and amino acidsequence (below, corresponding to SEQ ID NO: 56):

HindIIIAAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTG            M  D  M  R  V  L  A  Q  L  L  G  L  L  L  L  W  L  R  GCCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAA  R  C  D  I  Q  M  T  Q  S  P  S  S  L  S  A  S  V  G  D  R  V  TTCACTTGCCGGGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCCCI  T  C  R  A  S  Q  N  I  Y  K  Y  L  N  W  Y  Q  Q  R  P  G  K  ACTAAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATP  K  G  L  I  S  A  A  S  G  L  Q  S  G  V  P  S  R  F  S  G  S  GCTCAGGACAGATTTCACTCTCACCATCACCACATCTGCAACCTGAGATTTTGCAATTACTACTGTCAACS  G  T  D  F  T  L  T  I  T  S  L  Q  P  E  D  F  A  T  Y  Y  C  Q                                                        BsiWIAGAGTTACAGTCCCCCTCTCACTTTCGGCGCAAGGGACCAGGCATCAGAGATCAAACGTACGQ  S  Y  S  P  P  L  T  F  G  G  G  T  R  V  E  I  K  R  T

The amino acid sequence of the 8i10 Kappa LC variable region is asfollows, with specific domains identified below (CDR sequences definedaccording to Kabat methods):

M D M R V L A Q L L G L VK leader (SEQ ID NO: 57) L L L W L R G A R C DI Q M T Q S P S S L S FR1 (SEQ ID NO: 58) A S V G D R V T I T C R A S QN I Y K Y L N CDR1 (SEQ ID NO: 59) W Y Q Q R P G K A P K G FR2 (SEQ IDNO: 60) L I S A A S G L Q S CDR2 (SEQ ID NO: 61) G V P S R F S G S G S GFR3 (SEQ ID NO: 62) T D F T L T I T S L Q P E D F A T Y Y C Q Q S YS P PL T CDR3 (SEQ ID NO: 63) F G G G T R V E I K FR4 (SEQ ID NO: 64) R TStart of Kappa constant region

The following is an example of the Kappa LC variable region of 8i10cloned into the expression vector pcDNA3.1 which already contained theKappa LC constant region (upper polynucleotide sequence corresponds toSEQ ID NO: 65, lower polynucleotide sequence corresponds to SEQ ID NO:66, amino acid sequence corresponds to SEQ ID NO: 56 shown above). Basesin black represents pcDNA3.1 vector sequences, blue bases represent thecloned antibody sequences. The antibodies described herein have alsobeen cloned into the expression vector pCEP4.

The 8i10 Gamma HC variable region was cloned as a Hind III to Xho 1fragment, and is encoded the following polynucleotide sequences, and SEQID NO: 67 (top) and SEQ ID NO: 68 (bottom).

HindIII AAGCTTCCACCATGAAACACCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGCTGGGTTTCGAAGGTGGTACTTTGTGGACACCAAGAAGGAAGAGGACCACCGTCGAGGGTCGACCCACCTGTCCCAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGGGACAGGGTCCACGTTAACGTCCTCAGCCCGGGTCCTGACCACTTCGGAAGCCTCTGGGACTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGGCAGGGAGTGGACGTGACAGAGACCAAGCAGGTAGTCATTAATGATGACCTCGACCTAGGCCGAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAGTATCAGGGGTCCCTTCCCTGACCTCACCTAACCCAAATAGATAATGCCACCTTTGTGGTTCATCAATCCCTCCCTCAAGAGCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCGTTAGGGAGGGAGTTCTCGGCGCAGTGGTATAGTGTTCTGTGAAGGTTCTCAGTCCAGAGGCTGACGATGAGCTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGACTGCTACTCGAGACACTGGCGACGCCTTAGCCGGCAGATAAAGACACGCTCTCGCAGAA                                                       XhoIGTAGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCATCACCACCAATGACATAGGAACTGATGACCCCGGTCCCTTGGGACCAGTGGCAGAGCTC

The translation of the 8i10 Gamma HC is as follows, polynucleotidesequence (above, SEQ ID NO: 67, top) and amino acid sequence (below,corresponding to SEQ ID NO: 69):

HindIII AAGCTTCCACCATGAAACACCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGCTGGGTC            M  K  H  L  W  F  F  L  L  L  V  A  A  P  S  W  VCTGTCCCAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTG L  S  Q  V  Q  L  Q  E  S  G  P  G  L  V  K  P  S  E  T  LTCCCTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCGG S  L  T  C  T  V  S  G  S  S  I  S  N  Y  Y  W  S  W  I  RCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGGTTTATCTATTACGGTGGAAACACCAAG Q  S  P  G  K  G  L  E  W  I  G  F  I  Y  Y  G  G  N  T  KTACAATCCCTCCCTCAAGAGCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTC Y  N  P  S  L  K  S  R  V  T  I  S  Q  D  T  S  K  S  Q  VTCCCTGACGATGAGCTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCG S  L  T  M  S  S  V  T  A  A  E  S  A  V  Y  F  C  A  R  A                                                           XhoITCTTGTAGTGGTGGTTACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTC S  C  S  G  G  Y  C  I  L  D  Y  W  G  Q  G  T  L  V  T  V TCGAG  S

The amino acid sequence of the 8i10 Gamma HC is as follows with specificdomains identified below (CDR sequences defined according to Kabatmethods):

M K H L W F F L L L V A VH leader (SEQ ID NO:70) A P S W V L S Q V Q L QE S G P G L V FR1 (SEQ ID NO:71) K P S E T L S L T C T V S G S S I S N YY W S CDR1 (SEQ ID NO: 72) W I R Q S P G K G L E W FR2 (SEQ ID NO:73) IG F I Y Y G G N T K Y N P CDR2 (SEQ ID NO:74) S L K S R V T I S Q D T SK S Q FR3 (SEQ ID NO:75) V S L T M S S V T A A E S A V Y F C A R A S C SG G Y C I L D CDR3 (SEQ ID NO: 76) Y W G Q G T L V T V S FR4 (SEQ IDNO:77)

The following is an example of the Gamma HC variable region of 8i10cloned into the expression vector pcDNA3.1 which already contained theGamma HC constant region (upper polynucleotide sequence corresponds toSEQ ID NO: 78, lower polynucleotide sequence corresponds to SEQ ID NO:79, amino acid sequence corresponds to SEQ ID NO: 69 shown above). Basesin black represents pcDNA3.1 vector sequences, blue bases represent thecloned antibody sequences.

The framework 4 (FR4) region of the Gamma HC normally ends with twoserines (SS), so that the full framework 4 region should be W G Q G T LV T V S S (SEQ ID NO: 80). The accepting Xho 1 site and one additionalbase downstream of the Xho1 site in the vector, in which the Gamma HCconstant region that the Gamma HC variable regions are cloned, suppliesthe last bases, which encode this final amino acid of framework 4.However, the original vector did not adjust for the silent mutation madewhen the Xho1 site (CTCGAG, SEQ ID NO: 81) was created and contained an“A” nucleotide downstream of the Xho1 site, which caused an amino acidchange at the end of framework 4: a serine to arginine (S to R)substitution present in all the working Gamma HC clones. Thus, the fullframework 4 region reads W G Q G T L V T V S R (SEQ ID NO: 82). Futureconstructs are being created wherein the base downstream of the Xho 1site is a “C” nucleotide. Thus, the creation of the Xho 1 site used forcloning of the Gamma HC variable region sequences in alternativeembodiments is a silent mutation and restores the framework 4 amino acidsequence to its proper W G Q G T L V T V S S (SEQ ID NO: 80). This istrue for all M2 Gamma HC clones described herein.

Clone 21B15:

The Kappa LC variable region of the anti M2 clone 21B15 was cloned asHind III to BsiW1 fragment, and is encoded by the followingpolynucleotide sequences and SEQ ID NO: 83 and SEQ ID NO: 84:

HindIIIAAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTGCTTCGAAGGTGGTACCTGTACTCCCAGGAGCGAGTCGAGGACCCCGAGGACGATGAGACCGAGGCTCCACGCAGATGTGACATCCAGGTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCGTCTACACTGTAGGTCCACTGGGTCAGAGGTAGGAGGGACAGACGTAGACATCCTCTGTCTCAGTGGTAGACTTGCCGCGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCAGCAGAGACCAGGGAAAGCCCCTATGAACGGCGCGCTCAGTCTTGTAAATGTTCATAAATTTAACCATAGTCGTCTCTGGTCCCTTTCGGGGATAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGTCCCGGACTAGAGACGACGTAGGCCCAACGTTTCACCCCAGGGTAGTTCCAAGTCACCGTCACCTAGACCGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTCTGTCTAAAGTGAGAGTGGTAGTGGTCAGACGTTGGACTTCTAAAACGTTGAATGATGACAGTTGTCTCA                                                BsiWITACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGGTGGATATCAAACGTACGATGTCAGGGGGAGAGTGAAAGCCGCCTCCCTGGTCCCACCTATAGTTTGCATGC

The translation of the 21B15 Kappa LC variable region is as follows,polynucleotide sequence (above, SEQ ID NO: 83, top) and amino acidsequence (below, corresponding to SEQ ID NO: 56):

HindIIIAAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGT            M  D  M  R  V  L  A  Q  L  L  G  L  L  L  L  W  L  R  GGCCAGATGTGACATCCAGGTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC A  R  C  D  I  Q  V  T  Q  S  P  S  S  L  S  A  S  V  G  D  R  V  TATCACTTGCCGCGCGAGTCAGAACATTTACAAGTATTTAAATTGGTATCACCAGAGACCAGGGAAAGCC I  T  C  R  A  S  Q  N  I  Y  K  Y  L  N  W  Y  Q  Q  R  P  G  K  ACCTAAGGGCCTGATCTCTGCTGCATCCGGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGA P  K  G  L  I  S  A  A  S  G  L  Q  S  G  V  P  S  R  F  S  G  S  GTCTGGGACACATTTCACTCTCACCATCACCAGTCTCCAACCTGAAGATTTTCCAACTTACTACTGTCAA S  G  T  D  F  T  L  T  I  T  S  L  Q  P  E  D  F  A  T  Y  Y  C  Q                                                       BsiWICAGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAGGGTGGATATCAAACGTACG Q  S  Y  S  P  P  L  T  F  G  G  G  T  R  V  D  I  K  R  T

The amino acid sequence of the 21B15 Kappa LC variable region is asfollows, with specific domains identified below (CDR sequences definedaccording to Kabat methods):

M D M R V L A Q L L G L VK leader (SEQ ID NO: 57) L L L W L R G A R C DI Q V T Q S P S S L S FR1 (SEQ ID NO: 58) A S V G D R V T I T C R A S QN I Y K Y L N CDR1 (SEQ ID NO: 59) W Y Q Q R P G K A P K G FR2 (SEQ IDNO:60) L I S A A S G L Q S CDR2 (SEQ ID NO: 61) G V P S R F S G S G S GFR3 (SEQ ID NO: 62) T D F T L T I T S L Q P E D F A T Y Y C Q Q S Y S PP L T CDR3 (SEQ ID NO: 63) F G G G T R V D I K FR4 (SEQ ID NO: 64) R TStart of Kappa constant region

The primer used to clone the Kappa LC variable region extended across aregion of diversity and had wobble base position in its design. Thus, inthe framework 4 region a D or E amino acid could occur. In some cases,the amino acid in this position in the rescued antibody may not be theoriginal parental amino acid that was produced in the B cell. In mostkappa LCs the position is an E. Looking at the clone above (21B15) a Din framework 4 (D I K R T) (SEQ ID NO: 84) was observed. However,looking at the surrounding amino acids, this may have occurred as theresult of the primer and may be an artifact. The native antibody fromthe B cell may have had an E in this position.

The 21B15 Gamma HC variable region was cloned as a Hind III to Xho 1fragment, and is encoded by the following polynucleotide sequences andSEQ ID NO: 85 (top), and SEQ ID NO: 86 (bottom):

HindIII AAGCTTCCACCATGAAACACCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGCTGGGTCCTTCGAAGGTGGTACTTTGTGGACACCAAGAAGGAAGAGGACCACCGTCGAGGGTCGACCCAGGTGTCCCAGGTGCAATTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCACAGGGTCCACGTTAACGTCCTCAGCCCGCGTCCTGACCACTTCGCAAGCCTCTGGGACACGGTCACCTGCACTGTCTCTGGTTCGTCCATCAGTAATTACTACTGGAGCTGGATCCCGCAGTCCCAGTGGACGTGACAGAGACCAAGCAGGTAGTCATTAATGATGACCTCGACCTAGGCCGTCAGGCCAGGGAAGCGACTGGACTGGATTCGGTTTATCTATTACGCTGGAAACACCAAGTACAATCCCTGTCCCTTCCCTGACCTCACCTAACCCAAATAGATAATGCCACCTTTGTGGTTCATGTTAGGGACCCTCAAGAGCCGCGTCACCATATCACAAGACACTTCCAAGAGTCAGGTCTCCCTGACGATGAGGCAGTTCTCCGCGCAGTGGTATAGTGTTCTGTCAAGGTTCTCAGTCCAGAGGGACTGCTACTGCTCTGTGACCGCTGCGGAATCGGCCGTCTATTTCTGTGCGAGAGCGTCTTGTAGTGGTGGTTCGAGACACTGGCCACGCCTTAGCCGGCAGATAAAGACACGCTCTCGCAGAACATCACCACCAA                                           XhoIACTGTATCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCAGTGACATAGCAACTGATCACCCCGCTCCCTTGGGACCAGTGGCAGAGCTC

The translation of the 21B15 Gamma HC is as follows, polynucleotidesequence (above, SEQ ID NO: 87, top) and amino acid sequence (below,corresponding to SEQ ID NO: 69):

The amino acid sequence of the 21B15 Gamma HC is as follows, withspecific domains identified below (CDR sequences defined according toKabat methods):

M K H L W F F L L L V A VH leader (SEQ ID NO: 70) A P S W V L S Q V Q LQ E S G P G L V FR1 (SEQ ID NO: 71) N Y Y W S CDR1 (SEQ ID NO: 72) W I RQ S P G K G L E W FR2 (SEQ ID NO: 73) I G F I Y Y G G N T K Y N P CDR2(SEQ ID NO: 74) S L K S R V T I S Q D T S K S Q FR3 (SEQ ID NO: 75) V SL T M S S V T A A E S A V Y F C A R A S C S G G Y C I L D CDR3 (SEQ IDNO: 76) Y W G Q G T L V T V S FR4 (SEQ ID NO: 77)

Clone 23K12:

The Kappa LC variable region of the anti M2 clone 23K12 was cloned asHind III to BsiW1 fragment (see below), and is encoded by the followingpolynucleotide sequences SEQ ID NO: 88 (top) and SEQ ID NO: 89 (below).

HindIIIAAGCTTCCACCATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTACTCTGGCTCCGAGGTTCGAAGGTGGTACCTGTACTCCCAGGAGCGAGTCGAGGACCCCGAGGACGATGAGACCGAGGCTCCTGCCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACGGTCTACACTGTAGGTCTACTGGGTCAGAGGTAGGAGGGACAGACGTAGACATCCTCTGTCTCAGACCATCACTTGCCGGACAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGMAACCAGGGATGGTAGTGAACGGCCTGTTCAGTCTCGTAATCGTCGATAAATTTAACCATAGTCGTCTTTGGTCCCTAAGCCCCTAAACTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGTTCGGGGATTTGAGGACTAGATACGACGTAGGTCAAACGTTTCACCCCAGGGTAGTTCCAAGTCACCCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGGTCTGCAACCTGAAGATTTTGCAACCTACGTCACCTAGACCCTGTCTAAAGTGAGAGTGGTAGTCGCCAGACGTTGGACTTCTAAAACGTTGGATG                                                            BsiWITACTGTCAACAGAGTTACAGTATGCCTGCCTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACGATGACAGTTGTCTCAATGTCATACGGACGGAAACCGGTCCCCTGGTTCGACCTCTAGTTTGCATGC

The translation of the 23K12 Kappa LC variable region is as follows,polynucleotide sequence (above, SEQ ID NO: 90, top) and amino acidsequence (below, corresponding to SEQ ID NO: 91).

The amino acid sequence of the 23K12 Kappa LC variable region is asfollows, with specific domains identified below (CDR sequences definedaccording to Kabat methods):

M D M R V L A Q L L G L VK leader (SEQ ID NO: 57) L L L W L R G A R C DI Q M T Q S P S S L S FR1 (SEQ ID NO: 58) A S V G D R V T I T C R T S QS I S S Y L N CDR1 (SEQ ID NO: 92) W Y Q Q K P G K A P K L FR2 (SEQ IDNO: 93) L I Y A A S S L Q S G V P S R CDR2 (SEQ ID NO: 94) F S G S G S GT D F T L T FR3 (SEQ ID NO: 95) I S G L Q P E D F A T Y Y C Q Q S Y S MP A CDR3 (SEQ ID NO: 96) F G Q G T K L E I K FR4 (SEQ ID NO: 114) R TStart of Kappa LC constant region

The 23K12 Gamma HC variable region was cloned as a Hind III to Xho 1fragment, and is encoded by the following polynucleotide sequences andSEQ ID NO: 97 (top) and SEQ ID NO: 98 (bottom).

HindIIIAAGCTTCCACCATGGAGTTGGGGCTGTGCTGGGTTTTCCTTGTTGCTATTTTAAAAGGTGTCCAGTTTCGAAGGTGGTACCTCAACCCCGACACGACCCAAAAGGAACAACGATAAAATTTTCCACAGGTCAGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGAATCTCCTCACTCCACGTCGACCACCTCAGACCCCCTCCGAACCAGGTCGGACCCCCCAGGGACTCTTAGAGGAGTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGCACGTCGGAGACCTAAGTGGCAGTCATCGTTGATGTACTCAACCCAGGCGGTCCGAGGTCCCTTCCGGCTGGAGTGGGTCTCAGTTATTTATAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCACCGACCTCACCCAGAGTCAATAAATATCACCACCATCGTGTATGATGCGTCTGAGGCACTTCCCGTGATTCTCCTTCTCCAGAGACAACTCCAAGAACACAGTGTTTCTTCAAATGAACAGCCTGAGAGCCGCTAAGAGGAAGAGGTCTCTGTTGAGGTTCTTGTGTCACAAAGAAGTTTACTTGTCGGACTCTCGGCAGGACACGGCTGTGTATTACTGTGCGAGATGTCTGAGCAGGATGCGGGGTTACGGTTTAGACGTCTTCCTGTGCCGACACATAATGACACGCTCTACAGACTCGTCCTACGCCCCAATGCCAAATCTGCAGA                         XhoI GGGGCCAAGGGACCACGGTCACCGTCTCGAGCCCCGGTTCCCTGGTGCCAGTGGCAGAGCTC

The translation of the 23K12 Gamma HC variable region is as follows,polynucleotide sequence (above, SEQ ID NO: 99, top), and amino acidsequence (below, corresponding to SEQ ID NO: 100):

The amino acid sequence of the 23K12 Gamma HC variable region is asfollows, with specific domains identified below (CDR sequences definedaccording to Kabat methods):

M E L G L C W V F L V A VH leader (SEQ ID NO: 101) I L K G V Q C E V Q LV E S G G G L V FR1 (SEQ ID NO: 102) Q P G G S L R I S C A A S G F T V SS N Y M S CDR1 (SEQ ID NO: 103) W V R Q A P G K G L E W FR2 (SEQ ID NO:104) V S V I Y S G G S T Y Y A D CDR2 (SEQ ID NO: 105) S V K G R F S F SR D N S K N FR3 (SEQ ID NO: 106) T V F L Q M N S L R A E D T A V Y Y C AR C L S R M R G Y G L D V CDR3 (SEQ ID NO: 107) W G Q G T T V T V S FR4(SEQ ID NO: 108)

Example 3 Identification of Conserved Antibody Variable Regions

The amino acid sequences of the three antibody Kappa LC and Gamma HCvariable regions were aligned to identify conserved regions andresidues, as shown below.

Amino acid sequence alignment of the Kappa LC variable regions of thethree clones:

Amino acid sequence alignment of the Gamma HC variable regions of thethree clones:

Clones 8I10 and 21B15 came from two different donors, yet they have thesame exact Gamma HC and differ in the Kappa LC by only one amino acid atposition 4 in the framework 1 region (amino acids M versus V, seeabove), (excluding the D versus E wobble position in framework 4 of theKappa LC).

Sequence comparisons of the variable regions of the antibodies revealedthat the heavy chain of clone 8i10 was derived from germline sequenceIgHV4 and that the light chain was derived from the germline sequenceIgKV1.

Sequence comparisons of the variable regions of the antibodies revealedthat the heavy chain of clone 21 B15 was derived from germline sequenceIgHV4 and that the light chain was derived from the germline sequenceIgKV1.

Sequence comparisons of the variable regions of the antibodies revealedthat the heavy chain of clone 23K12 was derived from germline sequenceIgHV3 and that the light chain was derived from the germline sequenceIgKV1.

Example 4 Production and Characterization of M2 Antibodies

The antibodies described above were produced in milligram quantities bylarger scale transient transfections in 293 PEAK cells. Crudeun-purified antibody supernatants were used to examine antibody bindingto influenza A/Puerto Rico/8/1932 (PR8) virus on ELISA plates, and werecompared to the binding of the control antibody 14C2, which was alsoproduced by larger scale transient transfection. The anti-M2 recombinanthuman monoclonal antibodies bound to influenza while the controlantibody did not (FIG. 9).

Binding was also tested on MDCK cells infected with the PR8 virus (FIG.10). The control antibody 14C2 and the three anti M2E clones: 8I10,21B15 and 23K12, all showed specific binding to the M2 protein expressedon the surface of PR8-infected cells. No binding was observed onuninfected cells.

The antibodies were purified over protein A columns from thesupernatants. FACs analysis was performed using purified antibodies at aconcentration of 1 ug per ml to examine the binding of the antibodies totransiently transfected 293 PEAK cells expressing the M2 proteins on thecell surface. Binding was measured testing binding to mock transfectedcells and cells transiently transfected with the Influenza subtype H₃N₂,A/Vietnam/1203/2004 (VN1203), or A/Hong Kong/483/1997 HK483 M2 proteins.As a positive control the antibody 14C2 was used. Unstained andsecondary antibody alone controls helped determined background. Specificstaining for cells transfected with the M2 protein was observed for allthree clones. Furthermore, all three clones bound to the high pathstrains A/Vietnam/1203/2004 and A/Hong Kong/483/1997 M2 proteins verywell, whereas the positive control 14C2 which bound well to H₃N₂ M2protein, bound much weaker to the A/Vietnam/1203/2004 M2 protein and didnot bind the A/Hong Kong/483/1997 M2 protein. See FIG. 11.

Antibodies 21B15, 23K12, and 8I10 bound to the surface of 293-HEK cellsstably expressing the M2 protein, but not to vector transfected cells(see FIG. 1). In addition, binding of these antibodies was not competedby the presence of 5 mg/ml 24-mer M2 peptide, whereas the binding of thecontrol chimeric mouse V-region/human IgG1 kappa 14C2 antibody (hu14C2)generated against the linear M2 peptide was completely inhibited by theM2 peptide (see FIG. 1). These data confirm that these antibodies bindto conformational epitopes present in M2e expressed on the cell or virussurface, as opposed to the linear M2e peptide.

Example 5 Viral Binding of Human Anti-Influenza Monoclonal Antibodies

UV-inactivated influenza A virus (A/PR/8/34) (Applied Biotechnologies)was plated in 384-well MaxiSorp plates (Nunc) at 1.2 μg/ml in PBS, with25 ∥l/well, and was incubated at 4° C. overnight. The plates were thenwashed three times with PBS, and blocked with 1% Nonfat dry milk in PBS,50 μl/well, and then were incubated at room temp for 1 hr. After asecond wash with PBS, MAbs were added at the indicated concentrations intriplicate, and the plates were incubated at room temp for 1 hour. Afteranother wash with PBS, to each well was added 25 μl of a 1/5000 dilutionof horseradish peroxidase (HRP) conjugated goat anti-human IgG Fc(Pierce) in PBS/1% Milk, and the plates were left at room temp for 1 hr.After the final PBS wash, the HRP substrate 1-Step™ Ultra-TMB-ELISA(Pierce) was added at 25 μl/well, and the reaction proceeded in the darkat room temp. The assay was stopped with 25 μl/well 1N H₂SO₄, and lightabsorbance at 450 nm (A450) was read on a SpectroMax Plus plate reader.Data are normalized to the absorbance of MAb 8I10 binding at 10 μg/ml.Results are shown in FIGS. 2A and 2B.

Example 6 Binding of Human Anti-Influenza Monoclonal Antibodies toFull-Length M2 Variants

M2 variants (including those with a high pathology phenotype in vivo)were selected for analysis. See FIG. 3A for sequences.

M2 cDNA constructs were transiently transfected in HEK293 cells andanalyzed as follows: To analyze the transient transfectants by FACS,cells on 10 cm tissue culture plates were treated with 0.5 ml CellDissociation Buffer (Invitrogen), and harvested. Cells were washed inPBS containing 1% FBS, 0.2% NaN₃ (FACS buffer), and resuspended in 0.6ml FACS buffer supplemented with 100 μg/ml rabbit IgG. Each transfectantwas mixed with the indicated MAbs at 1 μg/ml in 0.2 ml FACS buffer, with5×10⁵ to 10⁶ cells per sample. Cells were washed three times with FACSbuffer, and each sample was resuspended in 0.1 ml containing 1 μg/mlalexafluor (AF) 647-anti human IgG H&L (Invitrogen). Cells were againwashed and flow cytometry was performed on a FACSCanto device(Becton-Dickenson). The data is expressed as a percentage of the meanfluorescence of the M2-D20 transient transfectant. Data for variantbinding are representative of 2 experiments. Data for alanine mutantsare average readouts from 3 separate experiments with standard error.Results are shown in FIGS. 3B and 3C.

Example 7 Alanine Scanning Mutagenesis to Evaluate M2 Binding

To evaluate the antibody binding sites, alanine was substituted atindividual amino acid positions as indicated by site-directedmutagenesis.

M2 cDNA constructs were transiently transfected in HEK293 cells andanalyzed as described above in Example 6. Results are shown in FIGS. 4Aand 4B. FIG. 8 shows that the epitope is in a highly conserved region ofthe amino terminus of the M2 polypeptide. As shown in FIGS. 4A, 4B andFIG. 8, the epitope includes the serine at position 2, the threonine atposition 5 and the glutamic acid at position 6 of the M2 polypeptide.

Example 8 Epitope Blocking

To determine whether the MAbs 8I10 and 23K12 bind to the same site, M2protein representing influenza strain A/HK/483/1997 sequence was stablyexpressed in the CHO (Chinese Hamster Ovary) cell line DG44. Cells weretreated with Cell Dissociation Buffer (Invitrogen), and harvested. Cellswere washed in PBS containing 1% FBS, 0.2% NaN₃ (FACS buffer), andresuspended at 10⁷ cells/ml in FACS buffer supplemented with 100 μg/mlrabbit IgG. The cells were pre-bound by either MAb (or the 2N9 control)at 10 μg/ml for 1 hr at 4° C., and were then washed with FACS buffer.Directly conjugated AF647-8I10 or -23K12 (labeled with the AlexaFluor®647 Protein Labeling kit (Invitrogen) was then used to stain the threepre-blocked cell samples at 1 μg/ml for 10⁶ cells per sample. Flowcytometric analyses proceeded as before with the FACSCanto. Data areaverage readouts from 3 separate experiments with standard error.Results are shown in FIG. 5.

Example 9 Binding of Human Anti-Influenza Monoclonal Antibodies to M2Variants and Truncated M2 Peptides

The cross reactivity of mAbs 8i10 and 23K12 to other M2 peptide variantswas assessed by ELISA. Peptide sequences are shown in FIGS. 6A and 6B.Additionally, a similar ELISA assay was used to determine bindingactivity to M2 truncated peptides.

In brief each peptide was coated at 2 μg/mL to a flat bottom 384 wellplate (Nunc) in 25 μL/well of PBS buffer overnight at 4° C. Plates werewashed three times and blocked with 1% Milk/PBS for one hour at roomtemperature. After washing three times, MAb titers were added andincubated for one hour at room temperature. Diluted HRP conjugated goatanti-human immunoglobulin FC specific (Pierce) was added to each wellafter washing three times. Plates were incubated for one hour at roomtemperature and washed three times. 1-Step™ Ultra-TMB-ELISA (Pierce) wasadded at 25 l/well, and the reaction proceeded in the dark at room temp.The assay was stopped with 25 μl/well 1N H₂SO₄, and light absorbance at450 nm (A450) was read on a SpectroMax Plus plate reader. Results areshown in FIGS. 6A and 6B.

Example 10 In Vivo Evaluation of the Ability of Human Anti-InfluenzaMonoclonal Antibodies to Protect From Lethal Viral Challenge

The ability of antibodies, 23K12 and 8I10, to protect mice from lethalviral challenge with a high path avian influenza strain was tested.

Female BALB/c mice were randomized into 5 groups of 10. One day prior(Day −1 (minus one)) and two days post infection (Day +2 (plus two), 200ug of antibody was given via 200 ul intra-peritoneal injection. On Day 0(zero), an approximate LD90 (lethal dose 90) of A/Vietnam/1203/04influenza virus, in a volume of 30 μl was given intra-nasally. Survivalrate was observed from Day 1 through Day 28 post-infection. Results areshown in FIG. 7.

OTHER EMBODIMENTS

Although specific embodiments of the invention have been describedherein for purposes of illustration, various modifications may be madewithout deviating from the spirit and scope of the invention.Accordingly, the invention is not limited except as by the appendedclaims.

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. An isolated fully human monoclonal antibody, wherein said monoclonalantibody has the following characteristics a) binds to an epitope in theextracellular domain of the matrix 2 ectodomain (M2e) polypeptide of aninfluenza virus; b) binds to influenza A infected cells; and c) binds toinfluenza A virus.
 2. The antibody of claim 1, wherein said antibody isisolated from a B-cell from a human donor.
 3. The antibody of claim 1,wherein said epitope is non-linear.
 4. The antibody of claim 1, whereinsaid epitope comprises the amino-terminal region of the M2e polypeptide.5. The antibody of claim 1, wherein said epitope comprises the aminoacid at position 2, 5, and 6 of the M2e polypeptide, wherein amino acidposition numbers are in accordance with SEQ ID NO:
 1. 6. The antibody ofclaim 4, wherein the amino acid at a) position 2 is a serine; b)position 5 is a threonine; and c) position 6 is a glutamic acid.
 7. Theantibody of claim 1, wherein said epitope wholly of partially includesthe amino acid sequence SLLTEV (SEQ ID NO: 42)
 8. The antibody of claim1, wherein said antibody is 8I10, 21B15 or 23K12.
 9. An antibody thatbinds the same epitope as 8I10, 21B15 or 23K12.
 10. An isolated fullyhuman monoclonal anti-M2e antibody or fragment thereof, wherein saidantibody comprises a variable heavy chain (V_(H)) region comprising CDR1and CDR2, wherein said region is encoded by a human IGHV4 or IGHV3 V_(H)germline sequence, or a nucleic acid sequence that is homologous to thesaid V_(H) germline gene sequence.
 11. The antibody of claim 10, whereinsaid nucleic acid sequence that is homologous to the germline sequenceis at least 90% homologous to said germline sequence.
 12. The antibodyof claim 10, wherein said antibody further comprises a variable lightchain (VL) region encoded by a human IGHK1 V_(L) germline gene sequence,or a nucleotide acid sequence that is homologous to the said V_(L)germline gene sequence.
 13. The antibody of claim 12, wherein saidnucleic acid sequence that is homologous to the IGHV1 V_(L) germlinesequence is at least 90% homologous to the said IGHV1 V_(L) germlinesequence.
 14. An isolated anti-matrix 2 ectodomain (M2e) antibody,wherein said antibody has a heavy chain with three CDRs comprising anamino acid sequence selected from the group consisting of the amino acidsequences of NYYWS (SEQ ID NO: 72), FIYYGGNTKYNPSLKS (SEQ ID NO: 74),ASCSGGYCILD (SEQ ID NO: 76), SNYMS (SEQ ID NO: 103), VIYSGGSTYYAD SVK(SEQ ID NO: 105), and CLSRMRGYGLDV (SEQ ID NO: 107) and a light chainwith three CDRs that include an amino acid sequence selected from thegroup consisting of the amino acid sequences of RASQNIYKYLN (SEQ ID NO:59), AASGLQS (SEQ ID NO: 61), QQSYSPPLT (SEQ ID NO: 63), RTSQSISSYLN(SEQ ID NO: 92), AASSLQSGVPSRF (SEQ ID NO: 94), and QQSYSMPA (SEQ ID NO:96).
 15. An isolated anti-matrix 2 ectodomain (M2e) antibody, whereinsaid antibody has a heavy chain with three CDRs comprising an amino acidsequence selected from the group consisting of the amino acid sequencesof NYYWS (SEQ ID NO: 72), FIYYGGNTKYNPSLKS (SEQ ID NO: 74), ASCSGGYCILD(SEQ ID NO: 76), SNYMS (SEQ ID NO: 103), VIYSGGSTYYADSVK (SEQ ID NO:105), and CLSRMRGYGLDV (SEQ ID NO: 107), wherein said antibody bindsM2e.
 16. An isolated anti-matrix 2 ectodomain (M2e) antibody, whereinsaid antibody has a light chain with three CDRs that include an aminoacid sequence selected from the group consisting of the amino acidsequences of RASQNIYKYLN (SEQ ID NO: 59), AASGLQS (SEQ ID NO: 61),QQSYSPPLT (SEQ ID NO: 63), RTSQSISSYLN (SEQ ID NO: 92), AASSLQSGVPSRF(SEQ ID NO: 94), and QQSYSMPA (SEQ ID NO: 96), wherein said antibodybinds M2e.
 17. An isolated anti-matrix 2 ectodomain (M2e) antibody,wherein said antibody has a heavy chain with three CDRs comprising anamino acid sequence selected from the group consisting of the amino acidsequences of NYYWS (SEQ ID NO: 72), FIYYGGNTKYNPSLKS (SEQ ID NO: 74),and ASCSGGYCILD (SEQ ID NO: 76), and a light chain with three CDRs thatinclude an amino acid sequence selected from the group consisting of theamino acid sequences of RASQNIYKYLN (SEQ ID NO: 59), AASGLQS (SEQ ID NO:61), and QQSYSPPLT (SEQ ID NO: 63).
 18. An isolated anti-matrix 2ectodomain (M2e) antibody, wherein said antibody comprises a V_(H) CDR1region comprising the amino acid sequence of NYYWS (SEQ ID NO: 72); aV_(H) CDR2 region comprising the amino acid sequence of FIYYGGNTKYNPSLKS(SEQ ID NO: 74); a V_(H) CDR3 region comprising the amino acid sequenceof ASCSGGYCILD (SEQ ID NO: 76); a V_(L) CDR1 region comprising the aminoacid sequence of RASQNIYKYLN (SEQ ID NO: 59); a V_(L) CDR2 regioncomprising the amino acid sequence of AASGLQS (SEQ ID NO: 61); a V_(L)CDR3 region comprising the amino acid sequence of and QQSYSPPLT (SEQ IDNO: 63).
 19. An isolated anti-matrix 2 ectodomain (M2e) antibody,wherein said antibody has a heavy chain with three CDRs comprising anamino acid sequence selected from the group consisting of the amino acidsequences of SNYMS (SEQ ID NO: 103), VIYSGGSTYYADSVK (SEQ ID NO: 105),and CLSRMRGYGLDV (SEQ ID NO: 107) and a light chain with three CDRs thatinclude an amino acid sequence selected from the group consisting of theamino acid sequences of RTSQSISSYLN (SEQ ID NO: 92), AASSLQSGVPSRF (SEQID NO: 94), and QQSYSMPA (SEQ ID NO: 96).
 20. An isolated anti-matrix 2ectodomain (M2e) antibody, wherein said antibody comprises a V_(H) CDR1region comprising the amino acid sequence of SNYMS (SEQ ID NO: 103); aV_(H) CDR2 region comprising the amino acid sequence of VIYSGGSTYYADSVK(SEQ ID NO: 105), a V_(H) CDR1 region comprising the amino acid sequenceof CLSRMRGYGLDV (SEQ ID NO: 107); a V_(L) CDR1 region comprising theamino acid sequence of RTSQSISSYLN (SEQ ID NO: 92); a V_(L) CDR2 regioncomprising the amino acid sequence of AASSLQSGVPSRF (SEQ ID NO: 94); anda V_(L) CDR3 region comprising the amino acid sequence of QQSYSMPA (SEQID NO: 96).
 21. An isolated anti-matrix 2 ectodomain (M2e) antibody orfragment thereof, wherein said antibody comprises: (a) a V_(H) CDR1region comprising the amino acid sequence of SEQ ID NO: 72 or 103; (b) aV_(H) CDR2 region comprising the amino acid sequence of SEQ ID NO: 74 or105; and (c) a V_(H) CDR3 region comprising the amino acid sequence ofSEQ ID NO: 76 or 107, wherein said antibody binds M2e.
 22. The antibodyof claim 21, wherein said antibody further comprises: (a) a V_(L) CDR1region comprising the amino acid sequence of SEQ ID NO: 59 or 92; (b) aV_(L) CDR2 region comprising the amino acid sequence of SEQ ID NO: 61 or94; and (c) a V_(L) CDR3 region comprising the amino acid sequence ofSEQ ID NO: 63 or
 96. 23. An isolated fully human monoclonal anti-matrix2 ectodomain (M2e) antibody comprising: a) a heavy chain sequencecomprising the amino acid sequence of SEQ ID NO: 44 and a light chainsequence comprising amino acid sequence SEQ ID NO: 46 or b) a heavychain sequence comprising the amino acid sequence of SEQ ID NO: 50 and alight chain sequence comprising amino acid sequence SEQ ID NO:
 52. 24. Acomposition comprising the antibody of claim
 1. 25. The composition ofclaim 24, further comprising an anti-viral drug, a viral entry inhibitoror a viral attachment inhibitor.
 26. The composition of claim 25,wherein said anti-viral drug is a neuraminidase inhibitor, a HAinhibitor, a sialic acid inhibitor or an M2 ion channel inhibitor. 27.The method of claim 26, wherein said M2 ion channel inhibitor isamantadine or rimantadine.
 28. The method of claim 26, wherein saidneuraminidase inhibitor zanamivir, or oseltamivir phosphate.
 29. Thecomposition of claim 24, further comprising a second anti-influenza Aantibody.
 30. The antibody of claim 1, wherein said antibody isoperably-linked to a therapeutic agent or a detectable label.
 31. Amethod for stimulating an immune response in a subject, comprisingadministering to the patient the composition of claim
 24. 32. A methodfor the treatment or prevention of an influenza virus infection in asubject, comprising administering to the subject the composition ofclaim
 24. 33. The method of claim 32, wherein the method furthercomprises administering an anti-viral drug, a viral entry inhibitor or aviral attachment inhibitor.
 34. The method of claim 33, wherein saidanti-viral drug is a neuraminidase inhibitor, a HA inhibitor, a sialicacid inhibitor or an M2 ion channel.
 35. The method of claim 34, whereinsaid M2 ion channel inhibitor is amantadine or rimantadine.
 36. Themethod of claim 34, wherein said neuraminidase inhibitor zanamivir, oroseltamivir phosphate.
 37. The composition of claim 32, furthercomprising a second anti-influenza A antibody.
 38. The method of claim32, wherein said antibody is administered prior to or after exposure toinfluenza virus.
 39. The method of claim 32, wherein said antibody isadministered at a dose sufficient to promote viral clearance oreliminate influenza A infected cells.
 40. A method for determining thepresence of a influenza virus infection in a patient, comprising thesteps of: (a) contacting a biological sample obtained from the patientwith the antibody according to claim 1; (b) detecting an amount of theantibody that binds to the biological sample; and (c) comparing theamount of antibody that binds to the biological sample to a controlvalue, and therefrom determining the presence of the influenza virus inthe patient.
 41. A diagnostic kit comprising the antibody according toclaim 1.